Preventing And/Or Treating Cardiovascular Disease And/Or Associated Heart Failure

ABSTRACT

Methods are provided for reducing copper values for, by way of example, treating, preventing or ameliorating tissue damage such as, for example, tissue damage that may be caused by (i) disorders of the heart muscle (for example, cardiomyopathy or myocarditis) such as idiopathic cardiomyopathy, metabolic cardiomyopathy which includes diabetic cardiomyopathy, alcoholic cardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy, and hypertensive cardiomyopathy, (ii) atheromatous disorders of the major blood vessels (macrovascular disease) such as the aorta, the coronary arteries, the carotid arteries, the cerebrovascular arteries, the renal arteries, the iliac arteries, the femoral arteries, and the popliteal arteries, (iii) drug induced, and metabolic (including hypertensive and/or diabetic disorders of small blood vessels (microvascular disease) such as the retinal arterioles, the glomerular arterioles, the vasa nervorum, cardiac arterioles, and associated capillary beds of the eye, the kidney, the heart, and the central and peripheral nervous systems, (iv) plaque rupture of atheromatous lesions of major blood vessels such as the aorta, the coronary arteries, the carotid arteries, the cerebrovascular arteries, the renal arteries, the iliac arteries, the fermoral arteries and the popliteal arteries, (v) diabetes or the complications of diabetes.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/221,298, filedSep. 7, 2005; which is a continuation of, and claims priority to, U.S.Ser. No. 10/388,213, filed Mar. 12, 2003; which in turn is a US 371National Stage application of, and claims priority to, International PCTApplication No. NZ/03/00042, filed Mar. 10, 2003, which claims thebenefit of New Zealand Provisional Patent Application Serial No. 517721,filed Mar. 8, 2002, New Zealand Provisional Patent Application SerialNo. 517725, file Mar. 11, 2002; and U.S. Provisional Patent ApplicationSer. No. 60/364,382, filed Mar. 12, 2002. The contents of each of whichare hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention concerns methods of treatment, prevention or ameliorationof a disease, disorder or condition in a mammal (hereafter “treating”),including, for example, a human being, having undesired copper levelsthat cause or lead to tissue damage. Treating of mammals includes those,for example, predisposed to copper-involved or -mediated free radicaldamage of tissue and/or to copper-involved or -mediated impairment ofnormal tissue stem cell responses. The invention has application interalia to diabetes-related and non-diabetes-related heart failure,macrovascular disease or damage, microvascular disease or damage, and/ortoxic (e.g., hypertensive) tissue and/or organ disease or damage(including such ailments as may, for example, be characterized by heartfailure, cardiomyopathy, myocardial infarction, and related arterial andorgan diseases) and to related compounds, compositions, formulations,uses, and procedures.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art, or relevant, to thepresently described or claimed inventions, or that any publication ordocument that is specifically or implicitly referenced is prior art.

Glucose is the primary source of energy for the human body. Absorbedfrom the intestine it is metabolized by energy production (by conversionto water and carbon dioxide), conversion to amino acids and proteins orketo-acids, and storage as glycogen. Glucose metabolism is regulated bycomplex orchestration of hormone activities. While all dietary sugarsare broken down into various carbohydrates, the most important isglucose, which is metabolized in nearly all cells of the body. Glucoseenters the cell by facilitated diffusion (glucose transport proteins).This facilitated transport is stimulated very rapidly and effectively byan insulin signal, pursuant to which glucose transport into muscle andadipose cells is increased up to twenty fold. After glucose istransported into the cytoplasm, insulin then directs the disposition ofit by conversion of glucose to glycogen, to pyruvate and lactate, and tofatty acids.

Diabetes mellitus is heterogeneous group of metabolic disorders,connected by raised plasma glucose concentration and disturbance ofglucose metabolism with resulting hyperglycemia. The hyperglycemia indiabetes mellitus generally results from defects in insulin secretion,insulin action, or both. Although its etiology has been clouded, theWorld Health Organization (WHO) has set forth a classification schemefor diabetes mellitus that includes type 1 diabetes mellitus, type 2diabetes mellitus, gestational diabetes, and other specific types ofdiabetes mellitus. Former terms like IDDM (insulin-dependent diabetesmellitus), NIDDM (non-insulin dependent diabetes mellitus), andjuvenile-onset diabetes mellitus or adult-onset diabetes mellitus are nolonger primarily used to describe those conditions.

The terms “insulin-dependent diabetes” (IDDM) or “juvenile-onsetdiabetes” previously encompassed what is now referred to as type 1diabetes. Type 1 diabetes results from an autoimmune destruction of theinsulin-secreting β-cells of the pancreas. There are several markers ofthis autoimmune destruction, detectable in body fluids and tissues,including islet cell autoantibodies, autoantibodies to insulin,autoantibodies to glutamic acid decarboxylase (GAD65), andautoantibodies to the tyrosine phosphatases IA-2 and IA-2β. Whilegenetic factors are strongly implicated, the concordance rate in twinstudies is under 50% and supports a role for environmental factors,which are said to include viral infections. The autoimmune processtypically begins many years before clinical detection and presentation.The rate of β-cell destruction is quite variable, being rapid in someindividuals (mainly infants and children) and usually slow in adults.

The terms “non-insulin-dependent diabetes mellitus” (NIDDM) or“adult-onset diabetes” previously encompassed what is now referred to astype 2 diabetes mellitus. The disease usually develops after 40 years ofage. It is much more common that type 1 diabetes and comprisesapproximately 90% of all individuals with diabetes. Type 2 patients areusually older at the onset of disease, and are characterized by varioussymptoms. Insulin concentrations are mostly increased but they can benormal or decreased. Obesity is common. Diet and exercise regimensleading to weight reduction can ameliorate hyperglycemia. Oralhypoglycemic drugs are also used in an effort to lower blood sugar.Nevertheless, insulin is sometimes required to correct hyperglycemia,particularly as patients grow older or as their β-cells fail.

Two groups of disorders may be said to typify type 2 diabetes mellitus.The first one is a decreased ability of insulin to act on peripheraltissues, usually referred to as “insulin resistance.” Insulin resistanceis defined as a decreased biological response to normal concentrationsof circulating insulin and represents the primary underlyingpathological process. The second is the dysfunction of pancreaticβ-cells, represented by the inability to produce sufficient amounts ofinsulin to overcome insulin resistance in the peripheral tissues.Eventually, insulin production can be insufficient to compensate for theinsulin resistance due to β-cell dysfunction. The common result is arelative deficiency of insulin. Data support the concept that insulinresistance is the primary defect, preceding the derangement of insulinsecretion. As with type 1 diabetes, the basis of the insulin resistanceand insulin secretion defects is believed to be a combination ofenvironmental and genetic factors.

Gestational diabetes mellitus is usually asymptomatic and notnecessarily life threatening to the mother. The condition is associatedwith an increased incidence of neonatal morbidity, neonatalhypoglycemia, macrosomia and jaundice. Even normal pregnancies areassociated with increasing insulin resistance, mostly in the second andthird trimesters. Euglycaemia is maintained by increasing insulinsecretion. In those women who are not able to increase the secretion ofinsulin, gestational diabetes develops. The pathophysiology ofgestational diabetes mellitus is not well known but is said to includefamily history of diabetes mellitus, obesity, complications in previouspregnancies and advanced maternal age.

Other specific types of diabetes mellitus are heterogeneous, with thefollowing representing the largest groups: genetic defects of β-cellfunction; genetic defects in insulin action; diseases of the exocrinepancreas (e.g., pancreatitis, traumalpancreatectomy, neoplasia, cysticfibrosis, hemochromatosis, and others); other endocrinopathies (e.g.,acromegaly, Cushing's syndrome, glucagonoma, pheochromocytoma,hyperthyroidism, somatostatinoma, aldosteronoma, and others); drug- orchemical-induced diabetes mellitus (e.g., from vacor (an acuterodenticide released in 1975 but withdrawn as a general-use pesticide in1979 because of severe toxicity, exposure to vacor causing destructionof the beta cells of the pancreas and diabetes mellitus in survivors),pentamidine, nicotinic acid, glucocorticoids, thyroid hormone,diazoxide, beta-adrenergic agonists, thiazides, phenyloin,alfa-interferon, and others); infection-induced diabetes mellitus (e.g.,from congenital rubella, cytomegalovirus, and others); rare forms ofimmune-mediated diabetes; and, other genetic syndromes sometimesassociated with diabetes (e.g., Down syndrome, Klinefelter's syndrome,Turner's syndrome, Wolfram syndrome, Friedreich's ataxia, Huntington'schorea, Lawrence-Moon Beidel syndrome, Myotonic dystrophy, PorphyriaPrader-Willi syndrome, and others). The etiology and pathophysiology arevery different, mostly complicated or connected to insulin secretion andaction derangement, as well as signal transduction inside the cellsdisarrangement. See “The Expert Committee on the Diagnosis andClassification of Diabetes Mellitus: Committee Report 2001,” AmericanDiabetes Association, Diabetes Care 1997; 20:1183-97 (revised 1999;republished January 2002); Lernmark A., “Type I Diabetes,” Clin. Chem.45 (8B): 1331-8 (1999); Lebowitz R E., “Type 2 Diabetes: An Overview,”Clin. Chem. 45 (8B): 1339-45 (1999). The vast majority of cases ofdiabetes fall into two broad etiopathogenetic categories, type 1diabetes (characterized by an absolute deficiency of insulin secretion)and the much more prevalent type 2 diabetes (characterized by acombination of resistance to insulin action and an inadequatecompensatory insulin secretory response), Sixteen million people in theUnited States are estimated to have diabetes, and more than 90% of thesepatients have type 2 diabetes. National Center for Health Statistics.Health United Stats. Washington, D.C.: Government Printing Office, 1998.The World Health Organization estimates that the number of diabeticadults will more than double globally, from 143 million in 1997 to 300million in 2025, largely because of dietary and other lifestyle factors.

Diabetes mellitus is a chronic condition characterized by the presenceof fasting hyperglycemia and the development of widespread prematureatherosclerosis. Patients with diabetes have increased morbidity andmortality due to cardiovascular diseases, especially coronary arterydisease. Vascular complications in diabetes may be classified asmicrovascular, affecting the retina, kidney and nerves andmacrovascular, predominantly affecting coronary, cerebrovascular andperipheral arterial circulation. Thus, the chronic hyperglycemia ofdiabetes is associated with long-term damage, dysfunction, and failureof various organs, especially the eyes, kidneys, nerves, heart, andblood vessels and long-term complications of diabetes includeretinopathy with potential loss of vision; nephropathy leading to renalfailure; peripheral neuropathy with risk of foot ulcers, amputation, andCharcot joints; and autonomic neuropathy causing gastrointestinal,genitourinary, and cardiovascular symptoms and sexual dysfunction.Glycation of tissue proteins and other macromolecules and excessproduction of polyol compounds from glucose are among the mechanismsthought to produce tissue damage from chronic hyperglycemia. Patientswith diabetes have an increased incidence of atheroscleroticcardiovascular, peripheral vascular, and cerebrovascular disease.Hypertension, abnormalities of lipoprotein metabolism, and periodontaldisease are often found in people with diabetes.

Chronic hyperglycemia results in hyperglycosylation of multiple proteinsand is the hallmark of diabetes. Hyperglycosylated proteins have alteredfunction resulting in a spectrum of effects. Epidemiological studieshave confirmed that hyperglycemia is the most important factor in theonset and progress of diabetes complications, both in insulin-dependentand non-insulin-dependent diabetes mellitus. Mechanisms connectinghyperglycemia with complications of long-term diabetes have beeninvestigated indicating the involvement of nonenzymatic glycationprocesses. The nonenzymatic glycation process in one in which glucose ischemically bound to amino groups of proteins, but without the help ofenzymes. It is a covalent reaction where, by means of N-glycosidebonding, sugar-protein complex is formed through a series of chemicalreactions described by Maillard. In Maillard reactions, sugar-proteincomplexes are formed (Amadori rearrangement) and represent an earlyproduct of nonenzymatic glycation and an intermediary that is aprecursor of later compounds. Numerous intermediary products are thenformed, followed by complex product polymerization reactions resultingin heterogeneous structures called advanced glycation end products(AGE). It was believed that the primary role in Maillard reactions wasexclusively played by high glucose concentration. However, recent datashow that, in spite of the fact that sugars are the main precursors ofAGE compounds, numerous intermediary metabolites includingα-oxoaldehydes also participate in nonenzymatic glycation reactions.Such intermediary products are generated during glycolysis(methylglyoxal) or in the polyolic pathway, and they can also be formedby autooxidation of carbohydrates (glyoxal). Alpha-oxoaldehydes modifyAGEs surprisingly fast, in contrast to classical Maillard reactionswhich are slower.

Glycation has both physiological and pathophysiological significance. Inphysiological conditions glycation can be detected in the aging process,and the reactions are significantly faster and more intensive withfrequently increased glucose concentrations. In diabetology theimportance of these processes is manifest in two essential issues, theeffect of protein glycation on the change of protein structure andfunction, and the use of glycated proteins level as a parameter ofintegrated glycemia. A classical example of nonenzymatic glycation isthe formation of glycated hemoglobin (HbA1c). The degree of nonenzymaticglycation being directly associated with blood glucose levels, thepercentage of HbA1c in diabetes can be very increased. HbA1c was thefirst studied glycated protein, but it was soon discovered that other,various structural and regulatory proteins, are also subject tononenzymatic glycation forming glycation end-products.

Protein modification with AGE is irreversible, there being no enzymes inthe organism able to hydrolyze AGE compounds, which consequentlyaccumulate during the life span of a protein on which they had wereformed. Examples include all types of collagen, albumin, basic myelinprotein, eye lens proteins, lipoproteins and nucleic acid. AGEs changethe function of many proteins and contribute to various latecomplications of diabetes mellitus. The major biological effect ofexcessive glycation include the inhibition of regulatory moleculebinding, crosslinking of glycated proteins, trapping of soluble proteinsby glycated extracellular matrix, decreased susceptibility toproteolysis, inactivation of enzymes, abnormalities of nucleic acidfunction, and increased immunogenicity in relation to immune complexesformation.

It has also been reported that AGEs progressively accumulate on thetissues and organs that develop chronic complications of diabetesmellitus like retinopathy, nephropathy, neuropathy and progressiveatherosclerosis. Immunohistochemical methods have demonstrated thepresence of different AGE compounds in glomeruli and tubuli cells inboth experimental and human diabetic nephropathy. The AGE role inatherosclerosis may also be significant. For instance, reticulated andirreversible low-density lipoprotein (LDL) from the circulation binds toAGE-modified collagen of blood vessel walls. In the majority of bloodvessels such reticular binding delays normal outflow of LDL particleswhich penetrate vessel walls and thus enhance the deposit of cholesterolin the intima. This is followed by an accelerated development ofatherosclerosis.

The level of AGE proteins reflects kinetic balance of two oppositeprocesses, the rate of AGE compound formation and the rate of theirdegradation by means of receptors. AGE receptors participate in theelimination and change of aged, reticular and denaturated molecules ofextracellular matrix as well as all other AGE molecules. However, indiabetes mellitus AGE protein accumulation may exceed the ability oftheir elimination due to chronic hyperglycemia and excessive glycation.AGE receptors were first detected on macrophage cells, and AGE proteinbinding to macrophage cell receptors is believed to cause a cascade ofevents in the homeostasis of blood vessel walls and their milieu bymediation of cytokines and tissue growth factors. At least fourdifferent AGE receptors have been described, among which two belong tothe group of receptor scavengers. One of them is very similar, if notidentical, to the receptor that internalizes altered LDL particles.Receptors on endothelium cells differ and these cell membrane sites bindAGE-ligands (denoted “RAGE” receptors). They belong to immunoglobulinreceptor family and are prevalent in tissues. Binding of AGE compoundsto RAGEs leads to cellular stress. It is not currently known whethervariations in AGE level explain differences in susceptibility to developcomplications, but it has been theorized that gene diversity in AGEreceptors could offer an explanation.

Hyperglycemia induces a large number of alterations in vascular tissuethat potentially promote accelerated atherosclerosis. Currently, inaddition to the nonenzymatic glycosylation of proteins and lipids, twoother major mechanisms have emerged that encompass most of thepathologic alterations observed in the vasculature of diabetic animalsand humans: oxidative stress, protein kinase C (PKC) activation.Importantly, these mechanisms are not independent. For example,hyperglycemia-induced oxidative stress promotes the formation of AGEsand PKC activation, and both type 1 and type 2 diabetes are independentrisk factors for coronary artery disease (CAD), stroke, and peripheralarterial disease. Schwartz C J, et al., “Pathogenesis of theatherosclerotic lesion. Implications for diabetes mellitus,” DiabetesCare 15:1156-1167 (1992); Stamler 3, et al., “Diabetes, other riskfactors, and 12-yr cardiovascular mortality for men screened in theMultiple Risk Factor Intervention Trial.” Diabetes Care 16:434-444(1993). Atherosclerosis accounts for virtually 80% of all deaths amongNorth American diabetic patients, compared with one-third of all deathsin the general North American population, and more than 75% of allhospitalizations for diabetic complications are attributable tocardiovascular disease. American Diabetes Association, “Consensusstatement: role of cardiovascular risk factors in prevention andtreatment of macrovascular disease in diabetes,” Diabetes Care 16:72-78(1993).

The decline in heart disease mortality in the general U.S. populationhas been attributed to the reduction in cardiovascular risk factors andimprovement in treatment of heart disease. However, patients withdiabetes have not experienced the reduction in age-adjusted heartdisease mortality that has been observed in nondiabetics, and anincrease in age-adjusted heart disease mortality has been reported indiabetic women. Gu K, et al., “Diabetes and decline in heart diseasemortality in U.S. adults,” JAMA 281:1291-1297 (1999). Studies have alsoshown that diabetic subjects have more extensive atherosclerosis of bothcoronary and cerebral vessels than age- and sex-matched nondiabeticcontrols. Robertson W B, Strong J P, “Atherosclerosis in persons withhypertension and diabetes mellitus,” Lab Invest 18:538-551 (1968). Ithas also been reported that diabetics have a greater number of involvedcoronary vessels and more diffuse distribution of atheroscleroticlesions. Waller B F, et al., “Status of the coronary arteries atnecropsy in diabetes mellitus with onset after age 30 years. Analysis of229 diabetic patients with and without clinical evidence of coronaryheart disease and comparison to 183 control subjects,” Am J Med69:498-506 (1980). Large studies comparing diabetics with matchedcontrols have also shown that diabetic patients with established CADundergoing cardiac catheterization for acute myocardial infarction,angioplasty, or coronary bypass have significantly more severe proximaland distal CAD. Granger C B, et al., “Outcome of patients with diabetesmellitus and acute myocardial infarction treated with thrombolyticagents. The Thrombolysis and Angioplasty in Myocardial Infarction (TAMI)Study Group,” J Am Coll Cardiol 21:920-925 (1993); Stein B, et al.,“Influence of diabetes mellitus on early and late outcome afterpercutaneous transluminal coronary angioplasty,” Circulation 91:979-989(1995); Barzilay J I, et al., “Coronary artery disease and coronaryartery bypass grafting in diabetic patients aged>or=65 years [reportfrom the Coronary Artery Surgery Study (CASS) Registry],” Am J Cardiol74:334-339 (1994)). Postmortem and angioscopic evidence also shows asignificant increase in plaque ulceration and thrombosis in diabeticpatients. Davies M J, et al., “Factors influencing the presence orabsence of acute coronary artery thrombi in sudden ischaemic death,” EurHeart J 10:203-208 (1989); Silva J A, et al. “Unstable angina. Acomparison of angioscopic findings between diabetic and nondiabeticpatients,” Circulation 92:1731-1736 (1995).

CAD is not confined to particular forms of diabetes, and is prevalent inboth type 1 and type 2 diabetes. In type 1 diabetes, an excess ofcardiovascular mortality is generally observed after the age of 30.Krolewski A S, et al., “Magnitude and determinants of coronary arterydisease in juvenile-onset, insulin-dependent diabetes mellitus,” Am JCardiol 59:750-755 (1987). CAD risk was reported in this study toincrease rapidly after age 40, and by age 55, 35% of men and women withtype 1 diabetes die of CAD, a rate of CAD mortality that far exceededthat observed in an age-matched nondiabetic cohort. Id. Diabeticnephropathy in type 1 diabetics also increases the prevalence of CAD.Nephropathy leads to accelerated accumulation of AGEs in the circulationand tissue and parallels the severity of renal functional impairment.Makita Z, et al., “Advanced glycosylation end products in patients withdiabetic nephropathy,” N Engl J Med 325:836-842 (1991). In diabeticpatients reaching end-stage renal disease, overall mortality has beenreported to be greater than in nondiabetic patients with end-stage renaldisease. The relative risk for age-specific death rate from myocardialinfarction among all diabetic patients during the first year of dialysisis reportedly 89-fold higher than that of the general population.Geerlings W, et al., “Combined report on regular dialysis andtransplantation in Europe, XXI,” Nephral Dial Transplant 6[Suppl 4:5-29(1991). It has also been reported that the most common cause of death indiabetic patients who have undergone renal transplantation is CAD,accounting for 40% of deaths in these patients. Lemmers M J, Barry J M,“Major role for arterial disease in morbidity and mortality after kidneytransplantation in diabetic recipients,” Diabetes Care 14:295-301(1991).

With regard to people with type 2 diabetes, CAD is the leading cause ofdeath, regardless of duration of diabetes. Stamler J, et al., “Diabetes,other risk factors, and 12-yr cardiovascular mortality for men screenedin the Multiple Risk Factor Intervention Trial,” Diabetes Care16:434-444 (1993); Donahue R P, Orchard T J, “Diabetes mellitus andmacrovascular complications. An epidemiological perspective,” DiabetesCare 15:1141-1155 (1992). The increased cardiovascular risk is said tobe particularly striking in women. Barrett-Connor E L, et al., “Why isdiabetes mellitus a stronger risk factor for fatal ischemic heartdisease in women than in men? The Rancho Bernardo Study,” JAMA265:627-631 (1991).

The degree and duration of hyperglycemia are the principal risk factorsfor microvascular complications in type 2 diabetes. The Diabetes Controland Complications Trial Research Group, “The effect of intensivetreatment of diabetes on the development and progression of long-termcomplications in insulin-dependent diabetes mellitus,” N Engl J Med329:977-986 (1993). However, it has also been said that there is noclear association between the extent or severity of macrovascularcomplications and the duration or severity of the diabetes, and anincreased prevalence of CAD is apparent in newly diagnosed type 2diabetes subjects has been reported. Uusitupa M, et al., “Prevalence ofcoronary heart disease, left ventricular failure and hypertension inmiddle-aged, newly diagnosed type 2 (non-insulin-dependent) diabeticsubjects,” Diabetologia 28:22-27 (1985). It has also been reported thateven impaired glucose tolerance carries an increased cardiovascular riskdespite minimal hyperglycemia. Fuller J H, et al.,“Coronary-heart-disease risk and impaired glucose tolerance. TheWhitehall study,” Lancet 1:1373-1376 (1980).

Insulin resistance is a common condition and, associated with geneticpredisposition, sedentary lifestyle, and aging, it is exacerbated andproduced by obesity. Thus, even in the absence of diabetes, insulinresistance is reportedly a major risk factor for CAD. Lempiainen P, etal., “Insulin resistance syndrome predicts coronary heart disease eventsin elderly nondiabetic men,” Circulation 100:123-128 (1999). Impairedinsulin action coupled with compensatory hyperinsulinemia leads to anumber of proatherogenic abnormalities referred to as insulin resistancesyndrome, and the association of insulin resistance with severalestablished atherogenic risk factors apparently promotes atherosclerosismany years before overt hyperglycemia ensues. Ferrannini E, et al.,“Insulin resistance in essential hypertension,” N Engl J Med 317:350-357(1987); Zavaroni I, et al., “Risk factors for coronary artery disease inhealthy persons with hyperinsulinemia and normal glucose tolerance,” NEngl J Med 320:702-706 (1989); Peiris A N, et al., “Adiposity, fatdistribution, and cardiovascular risk,” Ann Intern Med 110:867-872(1989); Reaven G M, “Role of insulin resistance in human disease(syndrome X): an expanded definition,” Annu Rev Med 44:121-131 (1993).

Dyslipidemia associated with insulin resistance entails elevatedvery-low-density lipoprotein (VLDL)-triglyceride levels, lowhigh-density lipoprotein (HDL) levels, delayed postprandial clearance oftriglyceride-rich lipoprotein remnants, and the presence of the veryatherogenic, small, dense LDL particles. Grundy S M,“Hypertriglyceridemia, atherogenic dyslipidemia, and the metabolicsyndrome,” Am J Cardiol 81:18B-25B (1998). This atherogenic lipoproteinphenotype is the most common lipoprotein abnormality seen in patientswith CAD and is said to impart a risk for CAD at least equal to that ofisolated moderate to severe hypercholesterolemia. Austin M A, et al.,“Atherogenic lipoprotein phenotype. A proposed genetic marker forcoronary heart disease risk,” Circulation 82:495-506 (1990).Insulin-resistant subjects also exhibit endothelial dysfunction and ahypercoagulable state, and chronic subclinical inflammation has emergedas part of the insulin-resistance syndrome. C-reactive protein, a markerof inflammation associated with cardiovascular events, is independentlyrelated to insulin sensitivity. Festa A, et al., “Chronic subclinicalinflammation as part of the insulin resistance syndrome: the InsulinResistance Atherosclerosis Study (IRAS),” Circulation 102:42-47 (2000).The proatherogenic metabolic risk factors in insulin-resistance subjectsworsen continuously across the spectrum of glucose tolerance. Meigs J B,et al., “Metabolic risk factors worsen continuously across the spectrumof nondiabetic glucose tolerance. The Framingham Offspring Study,” AnnIntern Med 128:524-533 (1998). Whether compensatory hyperinsulinemiapromotes atherosclerosis in insulin-resistant subjects is not clear.

The atherogenic risk factor profile observed in insulin-resistancepatients accounts for only a portion of the excess risk for CAD inpatients with type 2 diabetes, indicating that hyperglycemia itselfplays a central role in accelerating atherosclerosis in these patients.Thus, insulin-resistant individuals who go on to develop type 2 diabetesbecome exposed also to the atherogenic effects of hyperglycemia.Furthermore, while the threshold above which hyperglycemia becomesatherogenic is unknown, it may be in the range defined as impairedglucose tolerance. Gerstein H C, Yusuf S, “Dysglycaemia and risk ofcardiovascular disease,” Lancet 347:949-950 (1996). Variouspopulation-based studies in patients with type 2 diabetes are reportedto have shown a positive association between the degree of glycemiccontrol and CAD morbidity and mortality in middle-aged and elderly type2 diabetic subjects. Turner R C, et al., “Risk factors for coronaryartery disease in non-insulin dependent diabetes mellitus: UnitedKingdom Prospective Diabetes Study (UKPDS: 23),” BMJ316:823-828 (1998);Kuusisto Jr, et al., “NIDDM and its metabolic control predict coronaryheart disease in elderly subjects,” Diabetes 43:960-967 (1994); LaaksoM, “Hyperglycemia and cardiovascular disease in type 2 diabetes,”Diabetes 48:937-942 (1999).

The metabolic abnormalities associated with types 1 and 2 diabetes alsoresult in profound changes in the transport, composition, and metabolismof lipoproteins. Lipoprotein metabolism is said to be influenced byseveral factors including type of diabetes, glycemic control, obesity,insulin resistance, the presence of diabetic nephropathy, and geneticbackground. Ginsberg H N, “Lipoprotein physiology in nondiabetic anddiabetic states. Relationship to atherogenesis,” Diabetes Care14:839-855 (1991). Abnormalities in plasma lipoprotein concentrationsare commonly observed in diabetic individuals and reportedly contributeto the atherosclerotic process. The level of glycemic control is themajor determinant of lipoprotein levels in type 1 diabetic patients.Garg A, “Management of dyslipidemia in IDDM patients,” Diabetes Care17:224-234 (1994). In well- to moderately-controlled diabetes,lipoprotein levels are usually within the normal range, while in poorlycontrolled type 1 diabetic patients, triglycerides are markedlyelevated, LDL is modesty increased (usually when HbA_(1c) is greaterthan 11%), and HDL levels are decreased.

In contrast to type 1 diabetes, the pathophysiology of dyslipidemia intype 2 diabetes results from a complex relationship betweenhyperglycemia and the insulin-resistance state. The typical lipoproteinprofile associated with type 2 diabetes includes high triglycerides, lowHDL levels, and normal LDL levels, the most consistent change reportedlybeing an increase in VLDL-triglyceride levels. Syvanne M, Taskinen M R,“Lipids and lipoproteins as coronary risk factors innon-insulin-dependent diabetes mellitus,” Lancet 350:SI20-SI23 (1997);Ginsberg H N, “Diabetic dyslipidemia: basic mechanisms underlying thecommon hypertriglyceridemia and low HDL cholesterol levels,” Diabetes45[Suppl 3]:S27-S30 (1996). HDL levels are typically approximately 25%to 30% lower than in nondiabetic subjects and are commonly associatedwith other lipid and lipoprotein abnormalities, particularly hightriglyceride levels.

Hypertriglyceridemia in type 2 diabetes results from high fasting andpostprandial triglyceride-rich lipoproteins, especially VLDL. Type 2diabetic subjects with hypertriglyceridemia have both overproduction andimpaired catabolism of VLDL. Increased VLDL production is almostuniformly present in patients with type 2 diabetes andhypertriglyceridemia. Increased VLDL production in diabetes is aconsequence of an increase in free fatty acid mobilization (becausemaintenance of stored fat in adipose tissue depends on the suppressionof hormone-sensitive lipase by insulin) and high glucose levels. Becausefree fatty acid availability is a major determinant of VLDL productionby the liver, VLDL overproduction and hypertriglyceridemia occur.

The rest of the dyslipidemic phenotype that characterizes insulinresistance and type 2 diabetes (low HDL and small, dense LDL)—which hasbeen termed atherogenic lipoprotein phenotype (Austin M A, et al,“Atherogenic lipoprotein phenotype. A proposed genetic marker forcoronary heart disease risk,” Circulation 82:495-506 (1990))—followsonce VLDL secretion increases, mainly through the action of cholesterylester transfer protein and lipoprotein compositional changes that occurin plasma. Ginsberg H N, “Insulin resistance and cardiovasculardisease,” J Clin Invest 106:453-458 (2000). Increased fatty acid flux tothe liver also results in the production of large triglyceride-rich VLDLparticles because the size of VLDL is also mainly determined by theamount of triglyceride available. VLDL size is an important determinantof its metabolic fate and large triglyceride-rich VLDL particles may beless efficiently converted to LDL, thereby increasing direct removalfrom the circulation by non-LDL pathways. In addition, overproduction oflarge triglyceride-rich VLDL is said to be associated with theatherogenic small, dense LDL subclass.

In type 2 diabetic subjects with more severe hypertriglyceridemia, VLDLclearance by lipoprotein lipase (LPL)—the rate-limiting enzymeresponsible for the removal of plasma triglyceride-rich lipoproteins—isalso reported to be impaired. Syvanne M, Taskinen M R, “Lipids andlipoproteins as coronary risk factors in non-insulin-dependent diabetesmellitus,” Lancet 350:SI20-SI23 (1997). LPL requires insulin formaintenance of normal tissue levels, and its activity is low in patientswith poorly controlled type 2 diabetes. The result is enzymatic activityinsufficient to match the overproduction rate, with further accumulationof VLDL triglyceride.

Triglyceride concentrations are associated with premature CAD, andstudies have shown that triglyceride-rich lipoproteins play an importantrole in the progression of atherosclerosis. Hodis H N, “Myocardialischemia and lipoprotein lipase activity,” Circulation 102:1600-1601(2000). Furthermore, in contrast to the controversy regardinghypertriglyceridemia as a risk factor for coronary heart disease (CHD)in the nondiabetic population, several studies indicate that elevatedtriglyceride levels are independently associated with increased CHD riskin diabetic patients. Hypertriglyceridemia in diabetic patients oftencorrelates with LDL density and subclass (i.e., small, dense LDL) anddecreased levels of HDL₂, which appear to increase overall risksynergistically. Havel R J, Rapaport E, “Management of primaryhyperlipidemia,” N Engl J Med 332:1491-1498 (1995).

Characterized by increased VLDL production and impaired removal, it hasbeen reported that patients with type 2 diabetes exhibit excessivepostprandial lipemia and impaired remnant clearance. Exaggeratedpostprandial lipemia resulting from impaired remnant clearance is afactor in atherogenesis, involving endothelial dysfunction and enhancedoxidative stress. Karpe F, “Postprandial lipoprotein metabolism andatherosclerosis,” J Intern Med 246:341-355 (1999); Zilversmit D B,“Atherogenesis: a postprandial phenomenon,” Circulation 60:473-485(1979); Patsch J R, et al., “Relation of triglyceride metabolism andcoronary artery disease. Studies in the postprandial state,”Arterioscler Thromb 12:1336-1345 (1992); Plotnick G D, et al., “Effectof antioxidant vitamins on the transient impairment ofendothelium-dependent brachial artery vasoactivity following a singlehigh-fat meal,” JAMA 278:1682-1686 (1997). Postprandial lipemia consistsof a heterogeneous group of triglyceride-rich particles of differentcomposition and origin. Although 80% of the increase in postprandialtriglyceride levels is accounted for by chylomicrons (which carry alarge number of triglyceride molecules), the number of endogenous(liver-derived) VLDL constitutes over 90% of the triglyceride-richparticles in the postprandial state. Delayed VLDL clearance results inthe accumulation of partially catabolized VLDL remnants that are reducedin size and enriched in cholesteryl ester, and evidence is said toindicate that these small, cholesteryl ester-enriched VLDL particles areatherogenic.

As in nondiabetic subjects, low high-density lipoprotein (HDL) levelsare said to be powerful indicators of CHD in diabetic patients.Decreased HDL levels in diabetes result from decreased production andincreased catabolism of HDL and are closely related to the abnormalmetabolism of triglyceride-rich lipoproteins. In insulin-resistantpatients with or without overt type 2 diabetes, the composition of LDLparticles is altered, resulting in a preponderance of small,triglyceride-enriched and cholesterol-depleted particles (phenotype B).A preponderance of small, dense LDL particles is related to manycharacteristics of insulin-resistance syndrome, and in nondiabeticsubjects, LDL subclass phenotype B is associated with other componentsof insulin-resistance syndrome, including central obesity, hypertension,glucose intolerance, and hyperinsulinemia. Selby J V, et al., “LDLsubclass phenotypes and the insulin resistance syndrome in women,”Circulation 88:381-387 (1993); Reaven G M, et al., “Insulin resistanceand hyperinsulinemia in individuals with small, dense low densitylipoprotein particles,” Clin Invest 92:141-146 (1993); Haffner S M, etal., “LDL size in African Americans, Hispanics, and non-Hispanic whites:the insulin resistance atherosclerosis study,” Arterioscler Thromb VascBiol 19:2234-2240 (1999).

The formation of small, dense LDL in diabetes occurs in a similarfashion to the increased formation of small and dense HDL₃. Cholesterylester transfer protein mediates the exchange of triglyceride from VLDLfor cholesteryl ester in LDL. If sufficient LDL cholesteryl ester isreplaced by triglyceride from VLDL, then when the particle comes intocontact with hepatic lipase hydrolysis of newly acquired triglyceride inLDL and HDL by HTGL in turn decreases the size of LDL particles. PackardC J, Shepherd J, “Lipoprotein heterogeneity and apolipoprotein Bmetabolism,” Arterioscler Thromb Vase Biol 17:3542-3556 (1997). Small,dense LDL has also been said to be associated with CAD riskindependently of the absolute concentrations of LDL cholesterol or otherCAD risk factors, small, dense LDL particles being more susceptible tooxidative modification. Tribble D L, et al., “Oxidative susceptibilityof low density lipoprotein subfractions is related to their ubiquinol-10and alpha-tocopherol content,” Proc Natl Acad Sci USA 91:1183-1187(1994). They are also particularly prone to induce endothelialdysfunction. Anderson T J, et al., “Endothelium-dependent coronaryvasomotion relates to the susceptibility of LDL to oxidation in humans,”Circulation 93:1647-1650 (1996). In addition, there is enhanced arterialwall penetration by small LDL particles. Nielsen L B, “Transfer of lowdensity lipoprotein into the arterial wall and risk of atherosclerosis,”Atherosclerosis 123:1-15 (1996).

Glycosylation occurs both on the apoB and phospholipid components ofLDL, and is said to result in profound functional alternations in LDLclearance and susceptibility to oxidative modification, Bucala R, etal., “Identification of the major site of apolipoprotein B modificationby advanced glycosylation end products blocking uptake by the lowdensity lipoprotein receptor,” J Biol Chem 270:10828-10832 (1995);Bucala R, et al., “Lipid advanced glycosylation: pathway for lipidoxidation in vivo,” Proc Natl Acad Sci USA 90:6434-6438 (1993). Clinicalstudies have reportedly shown an increased level of AGEs on LDL obtainedfrom diabetics compared with healthy individuals. Bucala R, et al.,“Modification of low density lipoprotein by advanced glycation endproducts contributes to the dyslipidemia of diabetes and renalinsufficiency,” Proc Natl Acad Sci USA 91:9441-9445 (1994).Glycosylation of LDL apoB occurs mainly on a positively charged lysineresidue within the putative LDL receptor binding domain, which isessential for the recognition of LDL by the LDL receptor. Id. LDLglycosylation increases with glucose levels and impairs LDLreceptor-mediated LDL clearance.

Another atherogenic effect of glycation is to increase LDLsusceptibility to oxidative modification. Advanced glycosylation of anamine-containing phospholipid component of LDL is accompanied byprogressive oxidative modification of unsaturated fatty acid residues.Thus, glycation is said to also confer increased susceptibility of LDLto oxidative modification, which has been considered a critical step inits atherogenicity. Lyons T J, “Glycation and oxidation: a role in thepathogenesis of atherosclerosis,” Am J Cardiol 71:26B-31B (1993); BowieA, et al., “Glycosylated low density lipoprotein is more sensitive tooxidation: implications for the diabetic patient?,” Atherosclerosis102:63-67 (1993).

Cholesterol lowering using agents such as pravastatin have been reportedto reduce the absolute risk of coronary events for diabetic patients.Goldberg R B, et al., “Cardiovascular events and their reduction withpravastatin in diabetic and glucose-intolerant myocardial infarctionsurvivors with average cholesterol levels: subgroup analyses in theCholesterol and Recurrent Events (CARE) trial,” Circulation 98:2513-2519(1998). However, the absolute clinical benefit achieved by cholesterollowering may be greater in diabetic than in nondiabetic patients withCAD because diabetic patients have a higher absolute risk of recurrentCAD and higher case fatality rates, or because LDL cholesterol indiabetic patients is more atherogenic. Aronson D, et al., “Mechanismsdetermining course and outcome of diabetic patients who have had acutemyocardial infarction,” Ann Intern Med 126:296-306 (1997).

The American Diabetes Association recommendations for the management ofhyperlipidemia in patients with diabetes generally follow the guidelinesof the National Cholesterol Education Program with several differences.American Diabetes Association. Position statement. “Management ofdyslipidemia in adults with diabetes,” Diabetes Care 21:179-182 (1998).Non-pharmacologic strategies to treat dyslipidemia in diabetics includedietary modification (similar to those recommended by the NationalCholesterol Education Program), weight loss, physical exercise, andimproved glycemic control. Id. In patients with type 1 diabetes, optimalglycemic control should result in normal or below normal lipoproteinlevels and prevent the atherogenic state associated with lipoproteinglycosylation. Improved diabetic control in type 2 diabetes isbeneficial but not always associated with reversal of lipoproteinabnormalities.

Improved glycemic control using pharmacologic agents such assulfonylureas, insulin, metformin (N,N-dimethylimidodicarbonimidicdiamide hydrochloride), or thiazolidinediones can also help. Themagnitude of improvement in triglycerides generally correlates with thechange in glucose levels rather than the mode of therapy. However,agents that improve insulin sensitivity such as metformin andthiazolidinediones can also lead to lower triglycerides. In addition,“perfect” glycemic control is not attained in many type 2 diabeticpatients, and relatively recent publications have argued against therelevance of the traditional classification to primary and secondary CHDprevention in the setting of diabetes. Haffner S M, “Management ofdyslipidemia in adults with diabetes,” Diabetes Care 21:160-178 (1998);Haffner S M, et al. “Mortality from coronary heart disease in subjectswith type 2 diabetes and in nondiabetic subjects with and without priormyocardial infarction,” N Engl J Med 339:229-234 (1998). The rationalestems from both the high event rates in diabetic patients withoutclinical evidence of CAD (presumably because of the high rates ofsubclinical atherosclerosis), as well as the worse prognosis in diabeticpatients who have had a clinical event compared with nondiabeticsubjects, leading to the suggestion that LDL cholesterol should belowered to less than 100 mg per dL in diabetic subjects without priorCAD. Id.

Endothelial cells situated at the vessel wall-blood interfaceparticipate in a number of important homeostatic and cellular functionsthat protect from atherosclerosis and intraluminal thrombosis.Endothelial dysfunction can promote both the formation ofatherosclerotic plaques and the occurrence of acute events and, indiabetes, is said to entail profound perturbations in several criticalfunctions of the endothelium that contribute to the initiation andprogression of the atherosclerotic process, as well as to the occurrenceof clinical events. It is believed that diabetes results in weakenedintercellular junctions, and that AGEs diminish endothelial barrierfunction. The endothelial lining of the large arteries is of thecontinuous type characterized by tight junctions in the lateral borders,which restrict the movement of macromolecules from reaching thesubendothelial space.

Leukocyte adhesion to the vascular endothelium also contributes todiabetic complications. Among the earliest events in atherogenesis isthe binding of mononuclear leukocytes to the endothelium with subsequententry into the vessel wall. This is mediated through the expression ofinducible adhesion molecules on the endothelial cell surface.Hyperglycemia stimulates the expression of vascular cell adhesionmolecule-1 (VCAM-1) and E selectin. In addition, AGE interaction withthe AGE receptor has been reported to result in the induction ofoxidative stress and, consequently, of the transcription factorNF-kappaB and VCAM-1. Thus, early events in the atherosclerosis processin diabetes may be mediated through enhanced adhesive interactions ofmonocytes with the endothelial surface.

Impaired endothelium-dependent relaxation, which is mediated through therelease of endothelium-derived relaxing factor (EDRF), is alsoreportedly a consistent finding in animal models and in human diabetesand occurs in a variety of vascular beds, including the coronaryarteries. Impaired endothelium-dependent relaxation has beendemonstrated in both type 1 and type 2 diabetes in the absence ofclinical complications, while endothelium-independent vasodilation ispreserved, and impaired endothelium-dependent relaxation can bedemonstrated in insulin-resistant subjects with normal glucosetolerance. De Vriese A S, et al., “Endothelial dysfunction in diabetes,”Br J Pharmacal 130:963-974 (2000). Thus, hyperglycemia is recognized asthe primary mediator of diabetic endothelial dysfunction. Williams S B,et al., “Acute hyperglycemia attenuates endothelium-dependentvasodilation in humans in vivo,” Circulation 97:1695-1701 (1998).Similar to the mechanism of endothelial dysfunction observed inhypercholesterolemia, hyperglycemia-induced endothelial dysfunction isthought to result primarily from increased generation of oxygen freeradicals that inactivate EDRF. Insulin resistance is also said tocontribute to endothelial dysfunction in diabetic patients. Steinberg HO, et al., “Obesity/insulin resistance is associated with endothelialdysfunction. Implications for the syndrome of insulin resistance,” JClin Invest 97:2601-2610 (1996).

Diabetes is also said to be characterized by a variety of individualalterations in the coagulation and fibrinolytic systems that combine toproduce a prothrombotic state. These alterations include increasedplatelet functional behavior, increased levels of several coagulationcomponents, and impaired fibrinolysis. The coagulation and fibrinolyticsystems are said to be especially important in atherosclerosis becauseof the substantial contribution that mural thrombosis may make to thelater stages of plaque progression, and because thrombotic occlusionplays a vital role in the development of clinical events. In the vastmajority of cases, the fundamental mechanism in the development ofpotentially life-threatening events such as unstable angina ormyocardial infarction is thrombosis arising at sites of plaquedisruption.

Platelet hyperaggregability, including the presence of spontaneousplatelet aggregation and increased platelet aggregability induced byconventional stimuli, also increases the risk for cardiovascular events.Platelets from diabetic subjects exhibit enhanced adhesiveness andhyperaggregability, and shear-induced platelet adhesion and aggregationare also increased in diabetic patients. Knobler H, et al.,“Shear-induced platelet adhesion and aggregation on subendothelium areincreased in diabetic patients,” Thromb Res 90:181-190 (1998). vonWillebrand factor (vWF) is involved in the initial adhesion of plateletsto the subendothelium of injured vessel wall and is among the mostimportant adhesive molecules mediating hemostatic interactions betweenplatelets and vessel wall components. Synthesized and secreted byendothelial cells, high circulating levels of vWF are considered markersof endothelial dysfunction. In diabetic patients plasma concentrationsof vWF are elevated and are closely associated with the presence ofvascular complications and endothelial dysfunction. Stehouwer C D, etal., “Urinary albumin excretion, cardiovascular disease, and endothelialdysfunction in non-insulin-dependent diabetes mellitus,” Lancet340:319-323 (1992). Epidemiologic data have also demonstrated a relationbetween plasma vWF and insulin-resistance syndrome. Conlan M G, et al.,“Associations of factor VIII and von Willebrand factor with age, race,sex, and risk factors for atherosclerosis. The Atherosclerosis Risk inCommunities (ARIC) Study,” Thromb Haemost 70:380-385 (1993). Increasedplasma concentration of vWF has been shown to be predictive ofre-infarction and mortality in survivors of myocardial infarction, ofcardiac events in healthy people and in patients with angina pectoris.The European Concerted Action on Thrombosis study showed that vWFpredictability was not affected by the adjustment with other classicalcoronary risk factors such as body mass index, lipid disorders orsmoking. As vWF levels are dependent on the acute phase reaction likefibrinogen, and vWF correlates positively with fibrinogen or C-reactiveprotein levels, it has to be evaluated if vWF is a risk factorirrespective of fibrinogen level. In type 2 diabetic patients vWF levelsare higher in microalbuminuric patients. vWF is reportedly poorly or notat all related to insulin resistance.

Hyperactive platelets may form microaggregates leading to capillarymicroembolization. In patients with diabetes the resulting relativetissue hypoxia may in the long-term precede clinically detectablemicroangiopathy. It has been speculated that microembolization of thevasa vasorum of the large vessels by hyperactive platelets may also bethe initial event in the development of atherosclerosis. Secretion ofmitogenic, oxidative or vasoconstrictive substances by plateletsactivated in response to endothelial injury amplifies and acceleratesthe progression of atherosclerosis. Acute thrombotic events in thearterial circulation are also triggered by platelets.

The fibrinolytic system is natural defense against thrombosis. A balanceexists between plasminogen activators and inhibitors, and impairment ofthis balance can be caused either by diminished release of tissueplasminogen activator (t-PA) or increased levels of plasminogenactivator inhibitor 1 (PAI-1). PAI-1 is a serine protease inhibitor andevidence suggests that it is the major regulator of the fibrinolyticsystem. It binds and rapidly inhibits both single- and two-chain t-PAand urokinase. t-PA and PAI-1 rapidly form an inactive irreversiblecomplex. Abnormalities of the fibrinolytic system have been described inboth type 1 and type 2 diabetes. Impaired fibrinolysis, as described indiabetes type 2, is commonly accompanied by an increased plasma levelsof PAI-1 and by increased concentration oft-PA antigen, which reflectspredominantly t-PA/PAI-1 complexes. In type 1 diabetes results aremixed, and diminished, normal and enhanced fibrinolysis have all beenreported. In subjects with type 2 diabetes a variety of risk factors areindependently associated with impaired fibrinolysis: obesity,hypertension, dyslipidaemia, glucose intolerance, hyperinsulinaemia andinsulin resistance. These factors often tend to converge and numerousstudies have attempted to dissect out the independent contribution ofthe above risk factors in determining fibrinolytic activity in diabetes,but this task has been hampered by the complex relationship betweenthem. In non-diabetic subjects, insulin resistance is paralleled byincreased insulin and both correlate with triglyceride levels. Thus anyone or more of these variables may explain interrelationship with PAI-1.By contrast in type 2 diabetes, insulin resistance, insulinconcentration and triglyceride levels are less tightly interdependent inexplaining increased PAI-1. Impaired fibrinolysis not only predisposesto thrombotic events but also plays a role in the formation andprogression of atherosclerotic lesions. Increased synthesis of PAI-1 hasbeen demonstrated in atherosclerotic lesions. This may lead to fibrindeposition during lesion rupture, contributing to the progression of thelesion. PAI-1 within the lesion inhibits plasmin formation, which playsan important role in cleaving extracellular matrix proteins, directly orvia activation of metalloproteinases. This may lead to stabilization andfurther growth of atherosclerotic lesion. Changes in the fibrinolyticsystem also play an important role in microangiopathy. Urokinase andplasmin are activators of latent metalloproteinases, such ascollagenases, that are responsible for proteolysis of extracellularmatrix proteins. Increased PAI-1 may lead to basement membranethickening observed in microangiopathy.

A large body of evidence also indicates strong independent directcorrelation between high fibrinogen plasma levels and an increased riskof CAD. Fibrinogen levels are often increased in diabetes, and thiselevation is associated with poor glycemic control. Kannel W B, et al.,“Diabetes, fibrinogen, and risk of cardiovascular disease: theFramingham Experience,” Am Heart J 120:672-676 (1990). The intensity ofendogenous fibrinolysis depends on a dynamic equilibrium involvingplasminogen activators, primarily tissue-type plasminogen activator, andinhibitors. The principal physiologic inhibitor of tissue-typeplasminogen activator is plasminogen activator inhibitor-1 (PAI-1).Attenuated fibrinolysis caused by an increase of PM-1 activity has beenassociated with increased risk for myocardial infarction in patientswith established CAD. Kohler H P, Grant P J, “Plasminogen-activatorinhibitor type 1 and coronary artery disease,” N Engl J Med342:1792-1801 (2000). Reduced plasma fibrinolytic activity caused byincreased PAI-1 levels is a characteristic feature of insulin resistanceand hyperinsulinemia. Elevated concentrations of PAI-1 have beenrecognized consistently in the plasma of hyperinsulinemic type 2diabetics but occur also in normoglycemic insulin-resistant subjects.Juhan-Vague I, Alessi M C, “PAI-1, obesity, insulin resistance and riskof cardiovascular events,” Thromb Haemost 78:656-660 (1997). Theproduction of PAI-1 by adipose tissue has been demonstrated and could bean important contributor to the elevated plasma PAI-1 levels observed ininsulin-resistant patients. Alessi M C, et al., “Production ofplasminogen activator inhibitor 1 by human adipose tissue: possible linkbetween visceral fat accumulation and vascular disease,” Diabetes46:860-867 (1997). Hyperglycemia can also increase PAI-1 levels becauseit stimulates transcription of the PAI-1 gene through an effect on itspromoter region. Chen Y Q, et al., “Sp1 sites mediate activation of theplasminogen activator inhibitor-1 promoter by glucose in vascular smoothmuscle cells,” J Biol Chem 273:8225-8231 (1998). Although it is possiblethat some of the hemostatic abnormalities in diabetes are partly markersof underlying vascular disease rather than the primary abnormalities,the clotting and fibrinolytic profile of diabetic patients is said tobear a striking similarity to that of patients at high risk for futurecardiovascular events. The prothrombotic state in diabetes is said tohelp explain the observation that intracoronary thrombus formation ismore frequently found by angioscopic examination in diabetic patientswith unstable angina, and its clinical correlate, the higher risk ofadverse outcome, namely death, nonfatal infarction, or recurrentunstable angina. Silva J A, et al., “Unstable angina. A comparison ofangioscopic findings between diabetic and nondiabetic patients,”Circulation 92:1731-1736 (1995); Aronson D, et al., “Mechanismsdetermining course and outcome of diabetic patients who have had acutemyocardial infarction,” Ann Intern Med 126:296-306 (1997); Malmberg K,et al., “Impact of diabetes on long-term prognosis in patients withunstable angina and non-Q-wave myocardial infarction: results of theOASIS (Organization to Assess Strategies for Ischemic Syndromes)Registry,” Circulation 102:1014-1019 (2000); Calvin J E, et al., “Riskstratification in unstable angina. Prospective validation of theBraunwald classification,” JAMA 273:136-141 (1995). Thus, hyperglycemiainduces a large number of alterations in vascular tissue thatpotentially promote accelerated atherosclerosis. Acosta J, et al.,“Molecular basis for a link between complement and the vascularcomplications of diabetes,” Proc Natl Acad Sci USA 97:5450-5455 (2000).

Protein kinase C is also involved and the metabolic consequences ofhyperglycemia are said to be seen in cells in which glucose transport islargely independent of insulin. The resulting intracellularhyperglycemia has been implicated in the pathogenesis of diabeticcomplications through the activation of the PKC system. Ishii H, et al.,“Amelioration of vascular dysfunctions in diabetic rats by an oral PKCbeta inhibitor,” Science 272:728-731 (1996); Koya D, King G L, “Proteinkinase C activation and the development of diabetic complications,”Diabetes 47:859-866 (1998). High ambient glucose concentrations activatePKC by increasing the formation of diacylglycerol (DAG), the majorendogenous cellular cofactor for PKC activation, from glycolyticintermediates such as dihydroxy-acetone phosphate andglyceraldehyde-3-phosphate. The elevation of DAG and subsequentactivation of PKC in the vasculature can be maintained chronically. XiaP, et al., “Characterization of the mechanism for the chronic activationof diacylglycerol-protein kinase C pathway in diabetes andhypergalactosemia,” Diabetes 43:1122-1129 (1994).

PKC is a family of at least 12 isoforms of serine and threonine kinases.Although several PKC isoforms are reportedly expressed in vasculartissue, in the rat model of diabetes there is a preferential activationof PKC b2 in the aorta, heart, and retina, and PKC b1 in the glomeruli.Inoguchi T, et al., “Preferential elevation of protein kinase C isoformbeta II and diacylglycerol levels in the aorta and heart of diabeticrats: differential reversibility to glycemic control by islet celltransplantation,” Proc Natl Acad Sci USA 89:11059-11063 (1992); Koya D,et al., “Characterization of protein kinase C beta isoform activation onthe gene expression of transforming growth factor-beta, extracellularmatrix components, and prostanoids in the glomeruli of diabetic rats,” JClin Invest 100:115-126 (1997). The PKC system is ubiquitouslydistributed in cells and is involved in the transcription of severalgrowth factors and in signal transduction in response to growth factors.In vascular smooth muscle cells, PKC activation has been reported tomodulate growth rate, DNA synthesis, and growth factor receptorturnover. PKC activation increases the expression of transforming growthfactor-b (TGF-b), which is one of the most important growth factors,regulating extracellular matrix production by activating gene expressionof proteoglycans and collagen and decreasing the synthesis ofproteolytic enzymes that degrade matrix proteins. Increased expressionof TGF-b is thought to lead to thickening of capillary basementmembrane, one of the early structural abnormalities observed in almostall tissues in diabetes. PKC b selective inhibitor (LY333531) attenuatesglomerular expression of TGF-b and extracellular matrix proteins such asfibronectin and type IV collagen. Koya, supra; Koya D, et al.,“Amelioration of accelerated diabetic mesangial expansion by treatmentwith a PKC beta inhibitor in diabetic db/db mice, a rodent model fortype 2 diabetes,” FASEB J 14:439-447 (2000). Hyperglycemia-induced PKCactivation also results in increased platelet-derived growth factor-breceptor expression on smooth muscle cells and other vascular wall cells(e.g., endothelial cells, monocyte-macrophages). Inaba T, et al.,“Enhanced expression of platelet-derived growth factor-beta receptor byhigh glucose. Involvement of platelet-derived growth factor in diabeticangiopathy,” Diabetes 45:507-512 (1996).

Oxidative stress is widely invoked as a pathogenic mechanism foratherosclerosis. Among the sequelae of hyperglycemia, oxidative stresshas been suggested as a potential mechanism for acceleratedatherosclerosis. Baynes J W, Thorpe S R, “Role of oxidative stress indiabetic complications: a new perspective on an old paradigm,” Diabetes48:1-9 (1999). Importantly, there appears to be a strong pathogenic linkbetween hyperglycemia-induced oxidant stress and otherhyperglycemia-dependent mechanisms of vascular damage, namely AGEsformation and PKC activation), and hyperglycemia can increase oxidativestress through several pathways. A major mechanism appears to be thehyperglycemia-induced intracellular reactive oxygen species, produced bythe proton electromechanical gradient generated by the mitochondrialelectron transport chain and resulting in increased production ofsuperoxide. Nishikawa T, et al., “Normalizing mitochondrial superoxideproduction blocks three pathways of hyperglycaemic damage,” Nature404:787-790 (2000). Two other mechanisms have been proposed that mayexplain how hyperglycemia causes increased reactive oxygen speciesformation. One mechanism involves the transition metal-catalyzedautoxidation of free glucose, as described in cell-free systems. Throughthis mechanism, glucose itself initiates an autoxidative reaction andfree radical production yielding superoxide anion (O₂ ⁻) and hydrogenperoxide (H₂O₂). Wolff S P, “Diabetes mellitus and free radicals. Freeradicals, transition metals and oxidative stress in the aetiology ofdiabetes mellitus and complications,” Br Med Bull 49:642-652 (1993). Theother mechanism involves the transition metal-catalyzed autoxidation ofprotein-bound Amadori products, which yields superoxide and hydroxylradicals and highly reactive dicarbonyl compounds. Baynes J W, Thorpe SR, “Role of oxidative stress in diabetic complications: a newperspective on an old paradigm,” Diabetes 48:1-9 (1999).

There is also evidence that hyperglycemia may compromise naturalantioxidant defenses. Under normal circumstances, free radicals arerapidly eliminated by antioxidants such as reduced glutathione, vitaminC, and vitamin E. Reduced glutathione content, as well as reducedvitamin E, have been reported in diabetic patients. Yoshida K, et al.,“Weakened cellular scavenging activity against oxidative stress indiabetes mellitus: regulation of glutathione synthesis and efflux,”Diabetologia 38:201-210 (1995); Karpen C W, et al., “Production of12-hydroxyeicosatetraenoic acid and vitamin E status in platelets fromtype I human diabetic subjects,” Diabetes 34:526-531 (1985).

The interaction between AGE epitopes and the cell surface AGE receptorup-regulate oxidative stress response genes and release oxygen radicals.Thus, hyperglycemia simultaneously enhances both AGEs formation andoxidative stress, and the mutual facilitatory interactions betweenglycation and oxidation chemistry can contribute synergistically to theformation of AGEs, oxidative stress, and diabetic complications. Indeed,there are reportedly strong correlations between levels of glycoxidationproducts in skin collagen and the severity of diabetic retinal, renal,and vascular disease. Beisswenger P J, et al., “Increasedcollagen-linked pentosidine levels and advanced glycosylation endproducts in early diabetic nephropathy,” J Clin Invest 92:212-217(1993). Oxidative stress may also be involved in the activation ofDAG-PKC in vascular tissue. Nishikawa T, et al., “Normalizingmitochondrial superoxide production blocks three pathways ofhyperglycaemic damage,” Nature 404:787-790 (2000). Oxidants produced inthe setting of hyperglycemia can activate PKC. Konishi H, et al.,“Activation of protein kinase C by tyrosine phosphorylation in responseto H₂O₂ ,” Proc Natl Acad Sci USA 94:11233-11237 (1997).

The risk for congestive heart failure (CHF) and idiopathiccardiomyopathy is also said to be strongly increased in diabetes. KannelW B, et al., “Role of diabetes in congestive heart failure: theFramingham study,” Am J Cardiol 34:29-34 (1974); Shindler D M, et al.,“Diabetes mellitus, a predictor of morbidity and mortality in theStudies of Left Ventricular Dysfunction (SOLVD) Trials and Registry,” AmJ Cardiol 77:1017-1020 (1996); Ho K K, et al., “The epidemiology ofheart failure: the Framingham Study,” J Am Coll Cardiol 22:6A-13A(1993). Although data on the effect of diabetes on the prognosis ofpatients with CHF are limited, several studies implicate diabetes as anindependent predictor of poor prognosis in this setting. In the Studiesof Left Ventricular Dysfunction study, diabetes was an independentpredictor of morbidity and mortality in patients with symptomatic heartfailure, asymptomatic patients with an ejection fraction less than orequal to 35%, and in the registry population. Shindler, supra. Onereason for the poor prognosis in patients with both diabetes andischemic heart disease seems to be an enhanced myocardial dysfunctionleading to accelerated heart failure. Grundy S M, et al., “Diabetes andcardiovascular disease: a statement for healthcare professionals fromthe American Heart Association,” Circulation 100:1134-1146 (1999).

The cardiomyopathic process associated with diabetes mellitus manifestsinitially as diminished left ventricular compliance in the presence ofnormal left ventricular systolic function. Zarich S W, et al.,“Diastolic abnormalities in young asymptomatic diabetic patientsassessed by pulsed Doppler echocardiography,” J Am Coll Cardiol12:114-120 (1988); Paillole C, et al., “Prevalence and significance ofleft ventricular filling abnormalities determined by Dopplerechocardiography in young type I (insulin-dependent) diabetic patients,”Am J Cardiol 64:1010-1016 (1989); Mildenberger R R, et al., “Clinicallyunrecognized ventricular dysfunction in young diabetic patients,” J AmColl Cardiol 4:234-238 (1984). Diastolic abnormalities occur in 27% to69% of asymptomatic diabetic patients. A lower ejection fraction inresponse to dynamic exercise in the presence of a normal restingejection fraction has been demonstrated in several studies, indicatingthat contractile reserve is decreased in many asymptomatic patients withdiabetes. Mildenberger, supra; Shapiro L M, et al., “Left ventricularfunction in diabetes mellitus. II: Relation between clinical featuresand left ventricular function,” Br Heart J 45:129-132 (1981); Mustonen JN, et al., “Left ventricular systolic function in middle-aged patientswith diabetes mellitus,” Am J Cardiol 73:1202-1208 (1994). Systolicdysfunction may appear, usually in patients with long-standing diseasewho suffer from advanced microvascular complications. Racy D C, “Whichleft ventricular function is impaired earlier in the evolution ofdiabetic cardiomyopathy? An echocardiographic study of young type 1diabetic patients,” Diabetes Care 17:633-639 (1994). However, evensubclinical cardiomyopathy with reduced myocardial reserve may becomeclinically important in the presence of myocardial ischemia or withcoexistent uncontrolled hypertension. Stone P H, et al., “The effect ofdiabetes mellitus on prognosis and serial left ventricular functionafter acute myocardial infarction: contribution of both coronary diseaseand diastolic left ventricular dysfunction to the adverse prognosis. TheMILTS Study Group,” J Am Coll Cordial 14:49-57 (1989).

It is also understood that the coexistence of hypertension and diabetesexerts a particularly deleterious effect on the heart. The coexistenceof hypertension has been considered a major factor in the expression ofdiastolic dysfunction in diabetic patients. Grossman E, Messerli F H,“Diabetic and hypertensive heart disease,” Ann Intern Med 125:304-310(1996). In hypertensive subjects, diabetes is an important precursor ofCHF, with a greater relative risk in women than in men. Levy D, et al.,“The progression from hypertension to congestive heart failure,” JAMA275:1557-1562 (1996). The mechanisms responsible for the increased riskfor the development of CHF are not fully understood, but may be relatedin part to an exaggerated increase in left ventricular mass. Grossman E,et al., “Left ventricular mass in diabetes-hypertension,” Arch InternMed 152:1001-1004 (1992).

Obesity, which is characterized by insulin resistance andhyperinsulinemia, is also strongly correlated with increased leftventricular mass independent of age and blood pressure. Lauer M S, etal., “The impact of obesity on left ventricular mass and geometry. TheFramingham Heart Study,” JAMA 266:231-236 (1991). Furthermore, leftventricular mass in normotensive obese subjects is related more to theseverity of insulin resistance than to the obesity itself as expressedby the body mass index. Sasson Z, et al., “Insulin resistance is animportant determinant of left ventricular mass in the obese,”Circulation 88:1431-1436 (1993).

In hypertensive patients with normal glucose tolerance, who commonlyexhibit insulin resistance and hyperinsulinemia, left ventricular masshas been shown to correlate with the degree of insulin resistance. OhyaY, et al., “Hyperinsulinemia and left ventricular geometry in awork-site population in Japan,” Hypertension 27:729-734 (1996);Verdecchia P, et al., “Circulating insulin and insulin growth factor-1are independent determinants of left ventricular mass and geometry inessential hypertension,” Circulation 100:1802-1807 (1999). A similarassociation is also observed in nonhypertensive insulin-resistantsubjects. Marcus R, et al., “Sex-specific determinants of increased leftventricular mass in the Tecumseh Blood Pressure Study,” Circulation90:928-936 (1994).

Thus, hypertension, insulin resistance, hyperinsulinemia, and type 2diabetes are all reported to be commonly associated and result in a highrisk for cardiovascular complications. Reaven G M, Laws A, “Insulinresistance, compensatory hyperinsulinaemia, and coronary heart disease,”Diabetologia 37:948-952 (1994); Agewall S, et al., “Carotid artery wallintima-media thickness is associated with insulin-mediated glucosedisposal in men at high and low coronary risk,” Stroke 26:956-960(1995). Left ventricular mass is a strong predictor of cardiac andcerebrovascular morbidity independent of blood pressure or other riskfactors, as well as a powerful risk factor for the development ofsymptomatic CHF, and it is believed that the association between insulinresistance and left ventricular hypertrophy may contribute to theincrease risk of symptomatic CAD in insulin-resistant subjects.

Several reports have also focussed on metabolic abnormalities includingabnormal intracellular Ca²⁺” handling, defects in myocardial glucoseuse, and activation of PKC as possible explanations for the pathogenesisof diabetic cardiomyopathy. Additionally, there may also be a role forAGEs, discussed above, in the pathogenesis of diabetic cardiomyopathy.Diabetic patients have increased arterial stiffness compared withnondiabetic individuals and manifest diminished left ventricularcompliance at a young age. Several investigators have reported thatdiabetes has several features of accelerated aging at the tissue leveland at the level of collagen itself. Aging and diabetes mellitus areassociated with cross-linking and nonenzymatic glycosylation ofcollagen. This led to the concept that glycosylation could help toexplain the progressive cross-linking of collagen during normal agingand at an accelerated rate in diabetes, leading to changes in vasculartissue mechanical properties. Thus, disturbances of vascular and cardiacmechanical properties in diabetes may be caused by a common mechanism.Among the structural alterations associated with AGEs formation iscollagen-to-collagen cross-linking, which alters the structure andfunction of this protein, leading to tissue rigidity. Increased arterialstiffness in patients with diabetes is said to be strongly correlatedwith increased aorta and myocardial collagen advanced glycation.Airaksinen K E, et al., “Diminished arterial elasticity in diabetes:association with fluorescent advanced glycosylation end products incollagen,” Cardiovasc Res 27:942-945 (1993). Further evidence supportingthe AGE hypothesis is the observation that agents that specificallyinhibit AGE formation reportedly are useful to prevent the pathologicstiffening process of diabetes and aging. Norton G R, et al.,“Aminoguanidine prevents the decreased myocardial compliance produced bystreptozotocin-induced diabetes mellitus in rats,” Circulation93:1905-1912 (1996); Huijberts M S, et al., “Aminoguanidine treatmentincreases elasticity and decreases fluid filtration of large arteriesfrom diabetic rats,” J Clin Invest 92:1407-1411 (1993). For example,treatment of diabetic rats with aminoguanidine, an inhibitor of AGEformation, reportedly increased carotid artery compliance, decreasesaortic impedance, and prevented the decreased myocardial compliance. Id.

It has been attempted with greater or lesser efficacy topharmacologically influence the process of nonenzymatic glycation andAGE products formation using, in general, two approaches. The first isinhibition of the rearrangement from early to advanced glycationendproducts by means of hydrasine:aminoguanidine hydrochloride oranalogue. The second is the breaking of already existing AGE productswith substituted thiazolium salts. Pharmacologic activity ofaminoguanidine may render impossible or retard some of microvascularcomplications in animal model. Although the mechanism of aminoguanidineaction has not been completely understood, it may inhibit some stages ina series of chemical reactions leading to glycation end-productformation. In spite of the first encouraging results, clinical trials ofaminoguanidine in patients with type 2 diabetes mellitus have beensuspended due to adverse effects. See, for example, Brownlee M.,“Negative consequences of glycation,” Metabolism 49(suppl 1): 9-13(2000); Singh R, Barden A, Mori T, Beilin L., “Advanced glycationend-products: a review,” Diabetologia 44:129-146 (2001); Vlassara H,Bucala R, Striker L., “Pathogenic effects of AGEs: Biochemical,biologic, and clinical implications for diabetes and aging,” Lab Invest70:138-151 (1994); Lyons T, Jenkins A J., “Glycation, oxidation andlipoxidation in the development of the complications of diabetesmellitus: a ‘carbonyl stress’ hypothesis,” Diabetes Rev 5:365-391(1997).

As indicated herein, it is understood that diabetes mellitus is a majorsource of morbidity in developed countries. Among its co-morbidconditions, atherosclerosis is one of the most important. Since theavailability of insulin, up to three-quarters of all deaths amongdiabetics can be directly attributed to CAD. In patients with type 1diabetes, up to one third will die of CAD by the age of 50 years. Anumber of known risk factors for CAD, such as hypertension, centralobesity and dyslipidemia, are more common in diabetics than in thegeneral population. Thus diabetes represents a major contributing factorto the CAD burden in the developed world, and most of the excessattributed risk of CAD in diabetics cannot be readily quantified withthe use of traditional risk factors analysis. As indicated, the relationbetween hyperglycemia and CAD is the subject of debate because serumglucose does not consistently predict the existence of CAD. However,recent prospective data have clearly established a link between a markerfor chronic average glucose levels (HbA1c) and cardiovascular morbidityand mortality. There are established sequelae of hyperglycemia, such ascytotoxicity, increased extracellular matrix production and vasculardysfunction and all have been implicated in the pathogenesis ofdiabetes-induced vascular disease, and the formation of AGEs correlatedirectly with the vascular and renal complications of diabetes mellitus.As noted, patients with diabetes mellitus are particularly susceptibleto morbidity and mortality resulting from cardiovascular diseases,especially atherosclerosis, the progression of which is characterized byinfiltration of lipids into the vessel wall and the formation of fibroustissue called the atheromatous plaque. Clinical symptoms ofatherosclerosis do not usually occur until over half of the lumenbecomes obstructed (occluded) by the plaque, typically in the fifth andsixth decades of life. Consequently, studies on the role of plasmalipids in health and in the genesis of CHD have dominated research onCHD over the past several decades. Current positive evidence documentsthe premise that the following are important risk factors: familyhistory, a high plasma concentration of low-density lipoprotein (LDL)and a low concentration high density lipoprotein (HDL) cholesterol(separately as well as jointly), high plasma concentration of apoS (themajor protein fraction of the LDL particle), high plasma lipoprotein (a)(Lp(a)) concentration, high plasma fibrinogen concentration,hypertension, diabetes, obesity, increased plasma concentration ofhomocysteine (all these themselves have genetic determinants), highdietary fat intake, lack of exercise, stress, and smoking.

The pathogenesis of the atherosclerosis in diabetes mellitus is notentirely clear and conventional risk factors such as smoking, obesity,blood pressure and serum lipids fail to explain fully this excess risk.As noted, important features in the pathogenesis of atherosclerosisappear to include vascular endothelial injury, platelet adhesion andactivation, fibrin deposition, cellular proliferation, and low-densitylipoprotein cholesterol accumulation. Fibrin deposition is an invariablefeature in atherosclerotic lesions. Therefore, disturbances ofhaemostasis leading to accelerated fibrin formation (hypercoagulability)and delayed fibrin removal (impaired fibrinolysis) may contribute to thedevelopment of atherosclerosis. Hyperactive platelets,hypercoagulability and impaired fibrinolysis, as indicated above, alsopromote thrombosis formation at the site of ruptured atheroscleroticlesion and lead to final occlusion event in the progression ofatherosclerosis. Although platelet counts are generally normal inpatients with diabetes mellitus, multiple studies offer evidence ofenhanced activation or increased platelet activity, and an increase inplasma levels of vWF, which is important for the adhesion of plateletsto subendothelial structures, has been reported in diabetic patients.

In diabetes mellitus disturbances of haemostasis leading tohypercoagulability have been observed in numerous studies. Besidesaltered screening tests, alterations of several coagulation factors andinhibitors have been occasionally described. A problem encountered whenstudying the association between hypercoagulability and atherosclerosisis the number of laboratory tests proposed to detect hypercoagulabilityand the wide variability of such tests in a given subject. Results ofcohort studies have shown that among different coagulation factorsanalyzed, increased concentration of fibrinogen, factor VII and vWF havepredictive value for coronary atherosclerosis and can be considered asrisk factors for cardiovascular events. Increase in these factors couldparticipate in the pathogenesis of atherosclerosis, predominantly ofcoronary arteries. A relationship has been established between plasmaconcentration of fibrinogen, the quantity of fibrinogen and fibrinpresent in the vessel wall and the severity of atherosclerosis. Theseassociations are more pronounced in diabetic patients.

Factor VII is a vitamin K dependent protein synthesized in the liver. Itis the key enzyme in the initiation of blood coagulation. The NorthwickPark Heart Study and the PROCAM study have shown that there is apositive correlation between increased factor VII and cardiovascularmortality. Plasma concentration of factor VII is closely related toseveral environmental factors, mainly triglycerides and cholesterollevels. These associations are highly dependent on dietary intake. Anincrease in factor VII has been described in diabetes mellitus and ismore pronounced in those with microalbuminuria. Only limited data areavailable concerning the contributory role of insulin resistance toelevated factor VII. The relationship between factor VII and insulin andproinsulin have been described as very weak or present only in women.Factor VII which is influenced by the efficiency of the metabolism oftriglyceride-rich lipoproteins could in this way be modified in insulinresistance.

Hypercoagulability can also be judged from increased levels of markersof coagulation system activation, which reflect enhanced thrombingeneration. Prothrombin fragment 1+2 released when thrombin is formedfrom prothrombin is increased in diabetes. Once activated, thrombin israpidly inactivated by antithrombin, forming thrombin-antithrombincomplexes, which subsequently circulate and are removed by the liver.Multiple studies have documented elevated thrombin-antithrombincomplexes in diabetes. Fibrinopeptide A is released when fibrinogen isconverted to fibrin by thrombin. Thus, fibrinopeptide A levels areincreased during coagulation. Measurement of fibrinopeptide A indiabetes has yielded a variety of results, from elevated to normal.

hyperinsulinemia has also been associated with cardiovascular disease innon-diabetic subjects. In those with type 2 diabetes the extent ofhyperinsulinemia parallels plasma PAI-1 activity, and insulin has beenimplicated as a major physiological regulator of PAI-1. Despitepopulation correlations of insulin and PAI-1, and the effect of insulinon PAI-1 production in vitro, a direct effect of insulin on PAI-1 levelsin vivo in humans has not been shown, either with intravenous infusionof insulin or by an oral glucose load with the aim of producing portalhyperinsulinemia. Thus, in humans there is little evidence thatinterventions resulting in increased concentration of insulin in vivoincrease PAM. On the other hand reducing insulin levels and insulinresistance by exercise, weight loss and the drug metformin has beenshown to reduce PAI-1. In patients with type 2 diabetes approximately30% of fasting immunoreactive insulin concentration consists ofproinsulin-like molecules. The elevated levels of PAI-1 in thesesubjects may, therefore, be a consequence of precursor insulin ratherthan insulin itself.

Hyperglycemia is an additional risk factor for impaired fibrinolysis.Glucose can directly increase PAI-1 production in human endothelialcells. In patients with type 2 diabetes a significant correlationbetween glucose concentration and PAI-1 and has been observed. It hasbeen proposed that insulin resistance or hyperinsulinemia couldinfluence the synthesis of PAI-1 via effects on lipid metabolism. Inpatients with diabetes, dyslipidaemia, in particular high triglycerideand low high-density lipoprotein level, is common. Studies in vitro havereportedly demonstrated the effect of various lipoproteins on PAI-1synthesis. VLDLs from hypertriglycer-idemic patients increaseendothelial cell production of PAI-1 to a greater degree than that fromnormo-triglyceridaemic subjects. Oxidized LSLs also stimulateendothelial cell PAI-1 synthesis as does lipoprotein(a). Lipoprotein(a),LDSs, and HDLs also suppress t-PA secretion from human endothelial cellsin dose dependent manner.

In sum, there is significant laboratory evidence of chronic plateletactivation, enhanced coagulation and impaired fibrinolysis in patientswith diabetes mellitus. These disturbances of haemostasis favordevelopment of atherosclerosis and thrombosis in particularly ofcoronary arteries.

Metals are present naturally in body and many are essential for cells(e.g., Cu, Fe, Mn, Ni, Zn). However, all metals are toxic at higherconcentrations. One reason metals may become toxic is because they maycause oxidative stress, particularly redox active transition metals,which can take up or give off an electron (e.g., Fe2+/3+, Cu+/2+) cangive rise to free radicals that cause damage (Jones et al., “Evidencefor the generation of hydroxyl radicals from a chromium(V) intermediateisolated from the reaction of chromate with glutathione,” Biochim.Biophys. Acta 286: 652-655 (1991); Li, Y. and Trush, M. A. 1993. DNAdamage resulting from the oxidation of hydroquinone by copper: role fora Cu(II)/Cu(I) redox cycle and reactive oxygen generation,” Carcinogenes7: 1303-1311 (1993). Another reason why metals may be toxic is becausethey can replace other essential metals in or enzymes, disrupting thefunction of these molecules. Some metal ions (e.g., Hg+ and Cu+) arevery reactive to thiol groups and can interfere with protein structureand function.

As noted herein, humans subject to type 2 diabetes or abnormalities ofglucose mechanism are particularly at risk to the precursors of heartfailure, heart failure itself and a miscellany of other diseases of thearterial tree. It has been reported that in Western countries, more than50% of patients with type 2 diabetes die from the effects ofcardiovascular disease. See, Stamler et al., Diabetes Care 16:434-44(1993). It has also been reported that even lesser degrees of glucoseintolerance defined by a glucose tolerance test (impaired glucosetolerance, or “IGT”) still carry an increased risk of sudden death. See,Balkau et al., Lancet 354:1968-9 (1999). For a long time, it was assumedthat this reflected an increased incidence of coronary atherosclerosisand myocardial infarction in diabetic subjects. However, evidence ismounting that diabetes can cause a specific heart failure orcardiomyopathy in the absence of atherosclerotic coronary arterydisease.

Cardiac function is commonly assessed by measuring the ejectionfraction. A normal left ventricle ejects at least 50% of itsend-diastolic volume each beat. A patient with systolic heart failurecommonly has a left ventricular ejection fraction less than 30% with acompensatory increase in end-diastolic volume. Hemodynamic studiesconducted on diabetic subjects without overt congestive heart failurehave observed normal left ventricular systolic function (LV ejectionfraction) but abnormal diastolic function suggesting impaired leftventricular relaxation or filling. See, Regan et al., J. Clin. Invest.60:885-99 (1977). In a recent study, 60% of men with type 2 diabeteswithout clinically detectable heart disease were reported to haveabnounalities of diastolic filling as assessed by echocardiography. See,Poirier et al., Diabetes Care 24:5-10 (2001). Diagnosis may be made, forexample, by noninvasive measurements. In the absence of mitral stenosis,mitral diastolic blood flow measured by Doppler echocardiography is adirect measure of left ventricular filling. The most commonly usedmeasurement is the A/E ratio. Normal early diastolic filling is rapidand is characterized by an E-wave velocity of around 1 m/sec. Latediastolic filling due to atrial contraction is only a minor component,and the A-wave velocity is perhaps around 0.5 m/sec. This gives a normalA/E ratio of approximately 0.5. With diastolic dysfunction, earlydiastolic filling is impaired, atrial contraction increases tocompensate, and the A/E ratio increases to more than 2.0.

Treatment of diabetic cardiomyopathy is difficult and the options arelimited. Tight control of blood glucose levels might prevent or reversemyocardial failure, although this may be true only in the early stagesof ventricular failure. Angiotensin converting enzyme inhibitors such ascaptopril improve survival in heart failure particularly in patientswith severe systolic heart failure and the lowest ejection fractions.There are, however, various therapies for diabetic cardiomyopathy thatare not recommended. For example, inotropic drugs are designed toimprove the contraction of the failing heart. However, a heart with purediastolic dysfunction is already contracting normally and it is believedthat inotropic drugs will increase the risk of arrhythmias.Additionally, there appears to be no logical reason to use vasodilatordrugs that reduce after-load and improve the emptying of the ventriclebecause ejection fraction and end-diastolic volume are already normal.After-load reduction may even worsen cardiac function by creating adegree of outflow obstruction.

Diuretics are the mainstay of therapy for heart failure by controllingsalt and water retention and reducing filling pressures. However, theyare contraindicated in diastolic dysfunction where compromised cardiacpump function is dependent on high filling pressures to maintain cardiacoutput. Venodilator drugs such as the nitrates, which are very effectivein the management of systolic heart failure by reducing pre-load andfilling pressures, are understood to be poorly tolerated by patientswith diastolic heart failure. Ejection fraction and end-systolic volumeare often normal and any reduction in pre-load leads to a marked fall incardiac output. Finally, there is concern about the use of β-blockers inheart failure because of their potential to worsen pump function. Thereis also concern regarding the administration of β-blockers to patientswith diabetes who are treated with sulphonylurea drugs and insulin dueto a heightened risk of severe hypoglycaemia.

Thus, it will be understood that the mechanisms underlying variousdisorders of the heart, the macrovasculature, the microvasculature, andthe long-term complications of diabetes, including associated heartdiseases and conditions and long-term complications, are complex andhave long been studied without the discovery of clear, safe andeffective therapeutic interventions. There is a need for such therapies,which are described herein.

SUMMARY OF THE INVENTION

The heart is the most susceptible of all the body organs to prematureageing and free radical oxidative stress.

A high frequency of heart failure cardiomyopathy and macrovasculardisease in severely diabetic animals, for example, has been confirmed.It has also been discovered as described and claimed herein thattreatment with specific copper chelators and other agents (e.g., zincwhich prevents copper absorption) that decrease copper values and,preferably, do not lead to depletion states of other transition metals(e.g., iron, zinc and manganese), or essential metals, will benefit asignificant number and spectrum of the population, including for thosediseases, disorders, and/or conditions described above, whether or notattributable to diabetes or to any particular form of diabetes.

Preferably, treatment is preceded by a determination of the absence of acopper deficiency state or undesirably low copper values. Withoutwishing to be bound be bound by any particular theory or mechanism, itis believed that that copper values, particularly, for example, copper(II), that not bound internally within cells is available to mediatetogether with available reducing substances the generation of damagingfree radicals that have a role in both tissue damage and impairment ofstem cell mediated repair of such tissue.

By way of example, in relation to the normal myocardium tissue, it isbelieved that damage as a result of the presence of free radicals leadsto heart failure and/or cardiomyopathy in both diabetics andnondiabetics. The continued pressure of such free radicals is alsobelieved to impair stem cell mediated repair of the myocardium back toits normal healthy state. In respect of such damage and repairimpairment the present invention provides for a desired reduction inavailable free copper values as an appropriate preventive and/ortreatment approach.

By reference to available copper values in mammals (including humanbeings), those mammalian patients with a copper level that is “elevated”beyond that of the general population of such mammals can be identified.Reference herein to “elevated” in relation to the presence of coppervalues will include humans having at least about 10 mcg free copper/dLof serum when measured as discussed herein. A measurement of free copperequal to total plasma copper minus ceruloplasmin-bound copper can bemade using various procedures. A preferred procedure is disclosed in theMerck & Co datasheet (www.Merck.com) for SYPRINE® (trientinehydrochloride) capsules, a compound used for treatment of Wilson'sDisease, in which a 24 hour urinary copper analysis in is undertaken todetermine free cooper in the serum by calculating the difference betweenquantitatively determined total copper and ceruloplasmin-copper.Alternative names for trientine includeN,N′-Bis(2-aminoethyl)-1,2-ethanedi-amine; triethylenetetramine;1,8-diamino-3,6-diazaoetane; 3,6-diazaoctane-1,8-diamine;1,4,7,10-tetraazadecane; trien; TETA; TECZA and triene.

Without wishing to be bound by any particular theory or mechanism, it isbelieved that reduction in available free copper helps to preventmacrovascular, microvascular and/or toxic/metabolic diseases of the kindhereinafter exemplified and in tissue repair processes. This isirrespective of the glucose metabolism of the patient and is thusapplicable to diabetics and nondiabetics alike, as well as to those withand without impaired or abnormal glucose levels or metabolism.

Is also believed, again without wishing to be bound by any particulartheory or mechanism, that cardiovascular accumulation of redox-activetransition metal ions is responsible for many of the adverse outcomesand long term complications in diabetes. Under physiological conditions,injury to a target organ is sensed by distant stem cells, which migrateto the site of damage then undergo alternate stem cell differentiation.These events promote structural and functional repair. However, theaccumulation of redox-active transition metals, particularly copper incardiac or vascular tissues in subjects with diabetes is accompanied bya suppression of the normal tissue regeneration effected by themigration of stem cells. Elevated tissue levels of copper suppress thesenormal biological behaviors of such undifferentiated cells. Conditionsoccurring in the context of diabetes or impaired glucose tolerance, forexample, in which the suppression of normal stem cell responses cancause impairment of normal tissue responses, include cardiac failure,acute myocardial infarction, wound healing and ulceration, tissue damagecaused by infection, diabetic kidney damage, impaired cardiacregeneration, impaired vascular regeneration, and impaired regenerationof dependant organs.

Conditions in which therapy to lower copper values in diabetic patients(e.g., with IGT or type 2 diabetes mellitus) will prove beneficialinclude, for example, heart failure in the context of diabetes,myocardial infarction in the context of diabetes, wound healing andulceration in the context of diabetes, soft tissue damage resulting frominfection and occurring in the context of diabetes or impaired glucosetolerance, kidney damage occurring in the context of diabetes, impairedcardiac regeneration, impaired vascular regeneration, and impairedregeneration of dependant organs.

With regard to heart failure in the context of diabetes, significantregeneration of cardiac tissues can occur within a few days of cardiactransplantation. A likely mechanism is migration of stem cells fromextra-cardiac sites to the heart, with subsequent differentiation ofsuch cells into various specialized cardiac cells, including myocardial,endothelial and coronary vascular cells. Without wishing to be bound byany particular theory or mechanism, it is believed that copperaccumulation in cardiac tissues is likely to severely impair theseregenerative responses, and that there is a role for therapy, includingacute intravenous therapy, with transition a copper chelator in thetreatment of diabetic heart failure.

Regarding myocardial infarction (MI) in the context of diabetes, forexample, it is understood that MI is accompanied by proliferation ofcells in the ventricular myocardium. When MI occurs in the context ofdiabetes, the presence of elevated tissue levels of redox-activetransition metals suppresses normal stem cell responses, resulting inimpaired structural and functional repair of damaged tissues. It hasbeen reported that up to 20% of cells in the heart may be replaced bystem cell migration from extra-ventricular sites, as soon as four daysafter cardiac transplantation. It is believed that treatment of AMI inthe context of diabetes will be improved by, for example, acute (ifnecessary, parenteral) as well as by subsequent chronic administrationof chelators. Without wishing to be bound by any particular theory ormechanism, it is also believed that impairment of cardiac function indiabetes is characterized at least in part by a toxic effect ofaccumulated transition metals on tissue dynamics, resulting in impairedtissue regeneration caused in turn by suppression of normal stem cellresponses, which mediate physiological tissue regeneration by migrationto damaged tissue from external sites.

With regard to wound healing and ulceration in the context of diabetes,the processes of normal tissue repair require intervention of mobilizingstem cells, which effect repair of the various layers of blood vessels,for example. Without wishing to be bound by any particular theory ormechanism, it is believed that an accumulation of transition metals(particularly copper) in vascular tissues causes the impaired tissuebehaviour characteristic of diabetes, for example, including impairedwound repair following surgery or trauma, and the exaggerated tendencyto ulceration and poor healing of established ulcers. Without wishing tobe bound by any particular theory or mechanism, it is believed that thetreatment of diabetics with copper chelators before they undergosurgery, for example, or in the context of traumatic tissue damage, willbe of benefit. It is further believed that it is probable that surgeryin diabetics, for example, would have a better outcome if excesstransition metals in, for example, blood vessels, were removed orreduced prior to surgery. This may be accomplished, for example, oneither an acute basis (with parenteral therapy for example) or on a morechronic basis (with oral therapy for example) prior to actual surgery.

Regarding soft tissue damage resulting from infection and occurring inthe context of diabetes or impaired glucose tolerance, for example, andwithout wishing to be bound by any particular theory or mechanism, it isbelieved that the processes of normal tissue repair following infectionrequire intervention of mobilized stm cells that migrate to sites oftissue damage to effect tissue regeneration and repair, for example, ofthe various layers of blood vessels, and that repair of such tissuedamage will be impaired by suppressed stem cell responses, such as thosecaused by the build up of redox-active transition metals (particularlycopper) in tissues, for examples the walls of blood vessels. Treatmentwith a copper chelator or other agent to remove copper will improvethese conditions.

Regarding kidney damage occurring in the context of diabetes, againwithout wishing to be bound by any particular theory or mechanism, it isbelieved that impaired stem cell responses in the kidneys of diabeticscontribute to diabetic nephropathy and renal failure. Treatment ofdiabetics having kidney failure by administration of a copper chelatorwill improve organ regeneration by restoring normal tissue healing byallowing stem cells to migrate and differentiate normally. Furthermore,a reduction in extra-cellular copper values is also proposed to beadvantageous in the nondiabetic mammal and even in a mammal without aglucose mechanism abnormality, in that such lower levels will lead toone or both a reduction in copper-mediated tissue damage and improvedtissue repair by restoration of normal tissue stem cell responses.

Regarding impaired cardiac regeneration, again without wishing to bebound by any particular theory or mechanism, it is believed that copperaccumulation in cardiac tissues suppresses the normal tissueregeneration effected by the migration of stem cells from extra-cardiacsites to the heart, with subsequent differentiation of such cells intovarious specialised cardiac cells, including myocardial, endothelial,and coronary vascular cells. A reduction in extra-cellular copper valueswill reduce or ablate the impairment of tissue regeneration caused bythe suppression of normal stem cell responses.

With regard to impaired vascular regeneration, the processes of vascularregeneration is believed to require the intervention of mobilising stemcells, which effect repair of the various layers of blood vessels.Without wishing to be bound by any particular theory or mechanism, it isbelieved that an accumulation of transition metals (particularly copper)in vascular tissues causes impaired tissue regeneration by suppressingthe migration of undifferentiated stem cells and normal tissue stem cellresponses. A reduction in extracellular copper values is advantageous inthat such lower levels will lead to a reduction in the impairment ofvascular regeneration by restoration of normal tissue stem cellresponses.

As to impaired regeneration of dependant organs, it is believed, withoutwishing to be bound by any particular theory or mechanism, that anaccumulation of transition metals (particular copper) in the tissues ofthe dependant organs of the cardiovascular tree (e.g., retina, kidney,nerves, etc.) causes impaired tissue regeneration by suppressing themigration of stem cells and thereby the notuial biological behaviours ofsuch stem cells. A reduction in extra cellular copper values isadvantageous to reduce or ablate the impairment in tissue regenerationby restoration of normal tissue stem cell responses.

It is an object of the present invention to provide methods of treatmentand related methods, uses and pharmaceutical compositions thatameliorate, prevent or treat any one or more disease states of thecardiovascular tree (including the heart) and dependent organs (e.g.;retina, kidney, nerves, etc.) exacerbated by elevated non-intracellarfree copper values levels. Diseases of the cardiovascular tree anddiseases of dependent organs include, for example, but are not limitedto any one or more of:

disorders of the heart muscle (cardiomyopathy or myocarditis) such asidiopathic cardiomyopathy, metabolic cardiomyopathy which includesdiabetic cardiomyopathy, alcoholic cardiomyopathy, drug-inducedcardiomyopathy, ischemic cardiomyopathy, and hypertensivecardiomyopathy;

atheromatous disorders of the major blood vessels (macrovasculardisease) such as the aorta, the coronary arteries, the carotid arteries,the cerebrovascular arteries, the renal arteries, the iliac arteries,the femoral arteries, and the popliteal arteries;

toxic, drug-induced, and metabolic (including hypertensive and/ordiabetic disorders of small blood vessels (microvascular disease) suchas the retinal arterioles, the glomerular arterioles, the vasa nervorum,cardiac arterioles, and associated capillary beds of the eye, thekidney, the heart, and the central and peripheral nervous systems; and,

plaque rupture of atheromatous lesions of major blood vessels such asthe aorta, the coronary arteries, the carotid arteries, thecerebrovascular arteries, the renal arteries, the iliac arteries, thefemoral arteries and the popliteal arteries.

The present invention also relates to any such ailments and theirtreatment irrespective (unless otherwise stated) of any diabetic and/orglucose abnormality state of the mammalian patient.

Accordingly included within the categories of disease of patients thatare usefully be targeted by the procedures of the present invention are,for example, any one or more of the following non-exhaustive list:diabetic cardiomyopathy, diabetic acute coronary syndrome (e.g.;myocardial infarction—MI), diabetic hypertensive cardiomyopathy, acutecoronary syndrome associated with impaired glucose tolerance (IGT),acute coronary syndrome associated with impaired fasting glucose (IFG),hypertensive cardiomyopathy associated with IGT, hypertensivecardiomyopathy associated with IFG, ischaemic cardiomyopathy associatedwith IGT, ischaemic cardiomyopathy associated with IFG, ischaemiccardiomyopathy associated with coronary heart disease (CHD), acutecoronary syndrome not associated with any abnormality of the glucosemetabolism, hypertensive cardiomyopathy not associated with anyabnormality of the glucose metabolism, ischaemic cardiomyopathy notassociated with any abnormality of the glucose metabolism (irrespectiveof whether or not such ischaemic cardiomyopathy is associated withcoronary heart disease or not), and any one or more disease of thevascular tree including, by way of example, disease states of the aorta,carotid, cerebrovascular, coronary, renal, retinal, vasa nervorum,iliac, femoral, popliteal, arteriolar tree and capillary bed.

Without wishing to be bound by any particular theory or mechanism, it isbelieved that in the aforementioned diabetic states or glucosemetabolism abnormal states that diabetic complications in the distalregions of the arterial tree can be mediated by the regimen of thepresent invention whilst at the same time improving more proximalconditions.

With a nondiabetic patient the complications arising from an elevatedcopper values content of the whole body may be more proximal thandistal. Nonetheless mediation of and/or repair of such damage ispossible (including of or to the aorta, carotid, cerebrovascular,coronary, renal, retinal, vasa nervorum, iliac, femoral, popliteal,arteriolar tree and capillary bed) and it is believed will be improvedby the regimen of the present invention.

As used herein the term “diabetic” refers to a human being or othermammal suffering from type 2 diabetes or impaired glucose tolerance(IGT), or any other form of diabetes or impaired glucose metabolism inwhich removal of excess or undesired copper would be of value fortreatment.

The term “cardiomyopathy” as used herein and where the context so allowsincludes both cardiomyopathy and associated heart failure.

As used herein the terms “subjecting the patient” or “administering to”includes any active or passive mode of ensuring the in vivo presence ofthe active compound(s) or metabolite(s) irrespective of whether one ormore dosage to the mammal, patient or person is involved. Preferably themode of administration is oral. However, all other modes ofadministration (particularly parenteral, e.g., intravenous, intramuscular, etc.) are also contemplated.

As used herein, “therapeutically effective amount” refers to apredetermined amount of an agent that will or is calculated to achieve adesired response, for example, a therapeutic or preventative orameliorating response, for example, a biological or medical response ofa tissue, system, animal or human that is sought, for example, by aresearcher, veterinarian, medical doctor, or other clinician.

By “pharmaceutically acceptable” it is meant, for example, a carrier,diluent or excipient that is compatible with the other ingredients ofthe formulation and generally safe for administration to a recipientthereof or that does not cause an undesired adverse physical reactionupon administration.

As used herein, “mammal” has its usual meaning and includes primates(e.g.; humans and nonhumans primates), experimental animals (e.g.;rodents such as mice and rats), farm animals (such as cows, hogs, sheepand horses), and domestic animals (such as dogs and cats).

The term “elevated” has the meaning previously set forth whilst the term“normal” in respect of the copper values status of, for example, a humanpatient means, adopting the test referred to previously as having beendisclosed by Merck & Co Inc. is a patient having less than 10 mcg freecopper/dL of serum.

As used herein “copper deficient” means the diagnosis of copperdeficiency is usually made on the basis of low serum levels of copper(<65 μg/dL) and low ceruloplasmin levels (<18 mg/dL). Serum levels ofcopper may be elevated in pregnancy or stress conditions sinceceruloplasmin is an acute-phase reactant.

As used herein, the terms “treatment” or “treating” of a condition,disorder, and/or a disease in a mammal, means, where the context allows,(i) preventing the condition or disease, that is, avoiding one or moreclinical symptoms of the disease; (ii) inhibiting the condition ordisease, that is, arresting the development or progression of one ormore clinical symptoms; and/or (iii) relieving the condition or disease,that is, causing the regression of one or more clinical symptoms.

As used herein “associated with” simply means both circumstances existand should not be interpreted as meaning one necessarily is causallylinked to the other.

The term “chelatable copper” includes copper in any of its chelatableforms including different oxygen states such as copper (II). Accordinglythe term “copper values” (for example, elemental, salts, etc.) meanscopper in any appropriate form in the body available for such chelation(for example, in extracellular tissue and possibly bound to cellexteriors and/or collagen as opposed to intracellular tissue) and/orcapable of being reduced by other means (for example, zincadministration).

Some preferred chelators of copper values appropriate for mammalianadministration for treatment of one or more of the conditions, disordersand/or diseases herein include, for example (where appropriate as a saltsuch as, for example, a suitable calcium sodium salt to avoidhypocalcemia): trientine (triene), ethylenediaminetetraacetic acid(EDTA), diethylenetriaminetetraacetic acid (DPTA), 2,2,2 tetraminetetrahydrochloride (TETA), 2,3,2 tetramine tetrahydrochloride,D-peniciflamine (DPA), 1,4,8,11 tetraazacyclotretradecane (Cyclam),5,7,7′,12,14,14′ hexamethyl-1,4,8,11 tetraazacyclotretradecane (CyclamS), Sodium 2,3 dimercaptopropane-1-sulfonate (DMPS),N-acetylpenicillamine (NAPA), D-Penicillamine (PA),’ Desferroxamine,2,3-dimercaptopropanol (BAL), 2,3-dimercaptosuccinic acid (DMSA),trithiomolybdate, 3-7-Diazanonan-1,9-diamin (BE 6184),1,4,8,11-tetraazacyclotetradecane-, 1,4,8,11-tetraacetic acid,1,4,8,11-tetraazabicyclo[6.6.2]hexadecane,4,11-bis(N,N-diethyl-amidomethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane,4,11-bis(amidoethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane,melatonin, clioquinol, cuprizone, N,N′-diethyldithiocarbamate, zincacetate, zinc salts, bathocuproinedisulfonic acid,bathocuprinedisulfonate, neocuproine (2,9-dimethyl-1,10-phenanthroline),tetrathiomolybdate, trimetazidine, triethylene tetraminetetrahydrochloride, 2,3,2-tetraamine, pyridine-2,6-bis(thiocarboxylicacid) or pyrrolidine dithiocarbamate, tetraethylenepentamine,N,N,N′,N-tetrakis(2-pyridylemethyl)ethylenediamine,1,4,7,11-tetraazaundecane tetrahydrochloride, tetraethylenepentaminepentahydrochloride, D-Penicillamine (DPA), 1,10-orthophenanthroline,3,4-Dihydroxybenzoic acid, 2,2′-bicinchinonic acid, diamsar, 3,4′,5,trihydroxystilbene (resveratrol), mercaptodextran, o-phenanthroline,disulfuram (antabuse), sar, calcium trisodiumdiethylenetriaminepentaacetate (salt of cpd above), and methimazole(1-methyl-2-thiolimidazole).

In another aspect, one or more agents capable of decreasing the coppervalues content of the patient, if a chelator, has a preferentialaffinity for copper values over the values of other trace metals (suchas iron, zinc and/or manganese).

In yet another aspect, the preferential affinity for copper values issuch that copper excess of from 100% to 500% over that of a normalhealthy mammal of the species can be controlled to such normal levels orapproaching such normal levels without leading to depletion or excessivedecreases in such other transition metals as iron, zinc and/ormanganese. It is particularly desirable not to induce diseases of suchtransition metal deficiencies, for example, anemia.

Administration of any copper chelator (for example, trientine) can be bya variety of routes including parenteral and oral. With such agents adose rate for oral administration may be about 10 times that forparenteral administration. This will overcome any loweredbioavailablity. With trientine a suitable parenteral dose is about 120mg/day in man.

Accordingly where the chelator is trientine hydrochloride (andirrespective of excipients, diluents, carriers and vehicles), forexample, the dosage or dosages in a human patient, if parenteral, is toprovide about 120 mg/day, and if oral, about 1200 mg/day.

Alternatively (and/or additionally) the agent capable of reducing coppervalues is a zinc salt (preferably as a flavoured aqueous solution) ortrithiomolybdate (also a chelator). Suitable zinc salts include, forexample: zinc acetate; zinc chloride; zinc sulphate; zinc salts ofintermediates of the citric acid cycle, such as citrate, isocitrate,ketoglutarate, succinate, malate; and, zinc glucoante.

With the preferred chelators herein referred to, or others, and suitablesalts of zinc including those referenced herein, or others, it ispossible to selectively decrease the copper values in the body as awhole (without reaching depletion states for other transition metals)even though it is believed that there is little decrease in coppervalues in the intra cellular tissue. It is believed that the decrease isprimarily extra cellular (for example, interstites, on the exterior ofcells and/or on collagen).

In one aspect the present invention is a method of improving tissuerepair in a mammalian patient of damaged tissue selected from that ofthe myocardium, the vascular tree and organs dependent on the vasculartree, said method comprising or including the step of subjected thepatient to, and/or administering to the patient, an agent or agentseffective in lowering the copper values content of the patient's bodysufficient to improve tissue repair.

In another aspect, the patient is not suffering from Wilson's Diseaseyet has an elevated copper values content. In yet another aspect, thereis at least one copper values status determination.

In still another aspect, the agent is trientine or a trientine typecopper chelation agent.

Trientine hydrochloride may be administered at dosages or a dosage toprovide, if parenteral, at least about 120 mg/day in a human patient,and if oral, at least about 1200 mg/day in a human patient.

In one aspect, the patient is a human being suffering from type 2diabetes mellitus.

It is believed the improvement of the tissue repair arises from arestoration of, or substantial restoration, of normal tissue stem cellresponses, although there is no intent to be bound by this mechanism.

The agent(s) may be selected from, for example, trientine (triene),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminetetraaceticacid (DPTA), 2,2,2 tetramine tetrahydrochloride (TETA), 2,3,2 tetraminetetrahydrochloride, D-penicillamine (DPA), 1,4,8,11tetraazacyclotretradecane (Cyclam), 5,7,7%12,14,14′ hexamethyl-1,4,8,11tetraazacyclotretradecane (Cyclam S), Sodium 2,3dimercaptopropane-1-sulfonate (DMPS), N-acetylpenicillamine (NAPA),D-Penicillamine (PA),’ Desferroxamine, 2,3-dimercaptopropanol (BAL),2,3-dimercaptosuccinic acid (DMSA), trithiomolybdate,3-7-Diazanonan-1,9-diamin (BE 6184),1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid,1,4,8,11-tetraazabicyclo[6.6.2]hexadecane,4,11-bis(N,N-diethyl-amidomethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane,4,11-bis(amidoethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane,melatonin, clioquinol, cuprizone, N,N′-diethyldithiocarbamate, zincacetate, zinc salts, bathocuproinedisulfonic acid,bathocuprinedisulfonate, neocuproine (2,9-dimethyl-1,10-phenanthroline),tetrathiomolybdate, trimetazidine, triethylene tetraminetetrahydrochloride, 2,3,2-tetraamine, pyridine-2,6-bis(thiocarboxylicacid) or pyrrolidine dithiocarbamate, tetraethylenepentamine,N,N,N′,N-tetrakis(2-pyridylemethyl)ethylenediamine,1,4,7,11-tetraazaundecane tetrahydrochloride, tetraethylenepentaminepentahydrochloride, D-Penicillamine (DPA), 1,10-orthophenanthroline,3,4-Dihydroxybenzoic acid, 2,2′-bicinchinonic acid, diamsar, 3,4′,5,trihydroxystilbene (resveratrol), mercaptodextran, o-phenanthroline,disulfuram (antabuse), sar, calcium trisodiumdiethylenetriaminepentaacetate (salt of cpd above), and/or methimazole(1-methyl-2-thiolimidazole).

The agent (agents) may also be a zinc salt (zinc salts).

Damage to be ameliorated, treated, and/or prevented, may be, forexample, damage that has arisen from any one or more of: (i) disordersof the heart muscle (cardiomyopathy or myocarditis) such as idiopathiccardiomyopathy, metabolic cardiomyopathy which includes diabeticcardiomyopathy, alcoholic cardiomyopathy, drug-induced cardiomyopathy,ischemic cardiomyopathy, and hypertensive cardiomyopathy; (ii)atheromatous disorders of the major blood vessels (macrovasculardisease) such as the aorta, the coronary arteries, the carotid arteries,the cerebrovascular arteries, the renal arteries, the iliac arteries,the femoral arteries, and the popliteal arteries; (iii) toxic,drug-induced, and metabolic (including hypertensive and/or diabeticdisorders of small blood vessels (microvascular disease) such as theretinal arterioles, the glomerular arterioles, the vasa nervorum,cardiac arterioles, and associated capillary beds of the eye, thekidney, the heart, and the central and peripheral nervous systems; (iv)plaque rupture of atheromatous lesions of major blood vessels such asthe aorta, the coronary arteries, the carotid arteries, thecerebrovascular arteries, the renal arteries, the iliac arteries, thefermoral arteries and the popliteal arteries.

The patient may be suffering from and/or be predisposed to heartfailure.

The patient may be suffering from diabetes or impaired glucosemetabolism, for example, type 2 diabetes mellitus.

In another aspect the invention is the use of a compound (a) whichitself in vivo or (b) which has at least one metabolite in vivo which is(i) a copper chelator or (ii) otherwise reduces available copper valuesfor the production of a pharmaceutical composition or dosage unit ableto reduce the level of copper in a mammal thereby to elicit by alowering of copper values in a mammalian patient an improvement oftissue repair of damaged tissue selected from that of the myocardium,the vascular tree and organs dependent on the vascular tree.

The damage may be that which has arisen from a disease selected, forexample from the group: (i) disorders of the heart muscle(cardiomyopathy or myocarditis) such as idiopathic cardiomyopathy,metabolic cardiomyopathy which includes diabetic cardiomyopathy,alcoholic cardiomyopathy, drug-induced cardiomyopathy, ischemiccardiomyopathy, and hypertensive cardiomyopathy; (ii) atheromatousdisorders of the major blood vessels (macrovascular disease) such as theaorta, the coronary arteries, the carotid arteries, the cerebrovasculararteries, the renal arteries, the iliac arteries, the femoral arteries,and the popliteal arteries; (iii) toxic, drug-induced, and metabolic(including hypertensive and/or diabetic disorders of small blood vessels(microvascular disease) such as the retinal arterioles, the glomerulararterioles, the vasa nervorum, cardiac arterioles, and associatedcapillary beds of the eye, the kidney, the heart, and the central andperipheral nervous systems; (iv) plaque rupture of atheromatous lesionsof major blood vessels such as the aorta, the coronary arteries, thecarotid arteries, the cerebrovascular arteries, the renal arteries, theiliac arteries, the fermoral arteries and the popliteal arteries.

The compound is may be selected from, for example: trientine (triene),ethylenediaminetetraacetic acid (EDTA), diethylenetriaminetetraaceticacid (DPTA), 2,2,2 tetramine tetrahydrochloride (TETA), 2,3,2 tetraminetetrahydrochloride, D-penicillamine (DPA), 1,4,8,11tetraazacyclotretradecane (Cyclam), 5,7,7′,12,14,14′ hexamethyl-1,4,8,11tetraazacyclotretradecane (Cyclam S), Sodium 2,3dimercaptopropane-1-sulfonate (DMPS), N-acetylpenicillamine (NAPA),D-Penicillamine (PA),’ Desferroxamine, 2,3-dimercaptopropanol (BAL),2,3-dimercaptosuccinic acid (DMSA), trithiomolybdate,3-7-Diazanonan-1,9-diamin (BE 6184),1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid,1,4,8,11-tetraazabicyclo[6.6.2]hexadecane,4,11-bis(N,N-diethyl-amidomethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane,4,11-bis(amidoethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane,melatonin, clioquinol, cuprizone, N,N′-diethyldithiocarbamate, zincacetate, zinc salts, bathocuproinedisulfonic acid,bathocuprinedisulfonate, neocuproine (2,9-dimethyl-1,10-phenanthroline),tetrathiomolybdate, trimetazidine, triethylene tetraminetetrahydrochloride, 2,3,2-tetraamine, pyridine-2,6-bis(thiocarboxylicacid) or pyrrolidine dithiocarbamate, tetraethylenepentamine,N,N,N′,N-tetrakis(2-pyridylemethyl)ethylenediamine,1,4,7,11-tetraazaundecane tetrahydrochloride, tetraethylenepentaminepentahydrochloride, D-Penicillamine (DPA), 1,10-orthophenanthroline,3,4-Dihydroxybenzoic acid, 2,2′-bicinchinonic acid, diamsar, 3,4′,5,trihydroxystilbene (resveratrol), mercaptodextran, o-phenanthroline,disulfuram (antabuse), sar, calcium trisodium.diethylenetriaminepentaacetate (salt of cpd above), and methimazole(1-methyl-2-thiolimidazole).

Preferably the compound is trientine or a trientine-type copperchelation agent.

Preferably the use involves pharmaceutically acceptable excipients,diluents and/or carriers.

The invention is also a dosage unit resulting from the use.

In another aspect the invention is a method of treating a mammalianpatient (e.g.; a human being) at risk of developing, with suspected orwith actual tissue damage to the myocardium, the vascular tree and/ororgans dependent on the vascular tree, which method comprises orincludes the step of subjecting the patient mammal to and/oradministering to the patient mammal one or more agents capable ofdecreasing the copper values content of the patient thereby to betterenable tissue repair. In a related aspect, the patient does not haveWilson's Disease yet has elevated copper values. Preferably the agent(s)is (are) a chelator (chelators) of copper. It is also preferred but notrequired that the agent(s) has (have) an affinity for copper over thatof iron.

In still another aspect the invention is a method of treating amammalian patient (for example, a human being) at risk of developing,with suspected or with actual tissue disease to the myocardium, thevascular tree and/or organs dependent on the vascular tree, which methodcomprises or includes the steps of determining the copper status of thepatient, and if the copper status of a patient is elevated yet, forexample, the patient is not suffering from Wilson's Disease, subjectingthe patient to and/or administering to the patient one or more agentscapable of decreasing the patient's copper values content thereby tobetter enable tissue repair.

The method may involve continual or periodic evaluating or monitoring ofthe copper status of the patient.

The determination of the copper status can be by reference toextra-cellular copper values.

The decreasing of the patient's copper values content may be, but is notnecessarily, from an elevated status being that typical of the coppervalues status of a human patient suffering from type 2 diabetic mellitusor other disease, disorder or condition, for example, over that of a nonsufferer.

The method may include the step of diagnosing and/or evaluating ormonitoring glucose levels.

The method may include the step of diagnosing and/or evaluating ormonitoring postprandial glycemia.

The method may include the step of diagnosing and/or evaluating ormonitoring renal function.

The method may include the step of diagnosing and/or evaluating ormonitoring hypertension.

The method may include the step of diagnosing and/or evaluating ormonitoring insulin resistance.

The method may include the step of diagnosing and/or evaluating ormonitoring impaired glucose tolerance.

The method may include the step of diagnosing and/or evaluating ormonitoring obesity.

The method may include the step of diagnosing alcoholism.

The method may include the step of diagnosing and/or evaluating ormonitoring a glucose mechanism abnormality of the patient.

In one aspect, the abnormality is type 2 diabetes mellitus, IGT and/orIFG.

The method may also include the step of diagnosing and/or evaluating ormonitoring macrovascular, microvascular, toxic and/or metabolic damagein the patient.

Damage to be prevented, treated, or ameliorated can be damage resultingfrom or associated with any one or more of: (i) disorders of the heartmuscle (for example, cardiomyopathy or myocarditis) such as idiopathiccardiomyopathy, metabolic cardiomyopathy which includes diabeticcardiomyopathy, alcoholic cardiomyopathy, drug-induced cardiomyopathy,ischemic cardiomyopathy, and hypertensive cardiomyopathy; (ii)atheromatous disorders of the major blood vessels (macrovasculardisease) such as the aorta, the coronary arteries, the carotid arteries,the cerebrovascular arteries, the renal arteries, the iliac arteries,the femoral arteries, and the popliteal arteries; (iii) toxic,drug-induced, and metabolic (including hypertensive and/or diabeticdisorders of small blood vessels (microvascular disease) such as theretinal arterioles, the glomerular arterioles, the vasa nervorum,cardiac arterioles, and associated capillary beds of the eye, thekidney, the heart, and the central and peripheral nervous systems; (iv)plaque rupture of atheromatous lesions of major blood vessels such asthe aorta, the coronary arteries, the carotid arteries, thecerebrovascular arteries, the renal arteries, the iliac arteries, thefermoral arteries and the popliteal arteries.

In another aspect the present invention provides a method of treating amammalian patient (for example, a human being) at risk of developing,with suspected or with actual disease to the myocardium, the vasculartree and/or organs dependent therefrom, which method comprises orincludes the step of subjecting the patient to and/or administering tothe patient one or more agents capable of decreasing the copper valuescontent of the patient.

In another aspect the present invention provides a method of treating amammalian patient (for example, a human being) at risk of developing,with suspected or with actual disease to the myocardium, the vasculartree and/or organs dependent on the vascular tree, which methodcomprises or includes the steps of determining the copper status of thepatient, and if the copper status of a patient is undesirably elevatedor above that of a normal patient yet, for example, the patient is notsuffering from Wilson's Disease, subjecting the patient to and/oradministering to the patient one or more agents capable of decreasingthe patient's copper values content. The method may also involveperiodic or continual evaluating or monitoring of the copper status ofthe patient. The determination of the copper status is desirably byreference to extra cellular copper values.

Preferably the subjection or administration is with any one or more ofthe agents as herein referenced defined, preferred and/or exemplified.

In another aspect the present invention is the use of a compound (a)which itself in vivo or (b) which has at least one metabolite in vivowhich is a copper chelator or otherwise reduces available copper valuesfor the production of a pharmaceutical composition able to reduce thelevel of copper in a mammal (for example, in heart tissue and/or in thewalls of major blood vessels respectively) for the treatment (forexample, by repair of tissue resulting) of a disease (other than, forexample, Wilson's Disease) of any one or more of the kinds referred toherein.

In another aspect the invention is a method of improving tissue repairin a mammalian patient not suffering from Wilson's Disease yet having anelevated copper values body content, said method comprising or includingthe step of subjected the patient to, and/or administering to thepatient, an agent effective in lowering the copper values content of thepatient's body sufficient to improve tissue repair by restoration orsubstantially restoration of normal tissue stem cell responses. In stillanother aspect, there is at least one copper values statusdetermination.

In another aspect the present invention provides a method of treating amammal (for example, a human being) at risk of developing, withsuspected or with actual diabetic cardiomyopathy which comprises orincludes the step of subjecting the patient mammal to and/oradministering to the patient mammal one or more agents capable ofdecreasing the copper values content of the patient.

Such agent(s) may comprise or include copper chelators and/or mayinclude compounds or compositions otherwise capable of decreasing thecopper values content of the patient (for example, zinc (for example, asa suitable salt such as the gluconate salt) or tri thiomolybdate (also acopper chelator) which tend to prevent copper absorption by a patient).

The method may include an additional step or steps of evaluating ormonitoring the copper values of the patient prior to, simultaneouslywith and/or subsequent to the patient being subjected to or beingadministered with the agent(s).

The method may also include, for example, diagnosis of the patient as adiabetic.

In another aspect the present invention provides a method of treating amammal (for example, a human being) at risk of developing, withsuspected or with actual diabetic acute myocardial infarction whichcomprises or includes the step of subjecting the patient mammal toand/or administering to the patient mammal one or more agents capable ofdecreasing the copper values content of the patient. Such agent(s) maycomprise or include copper chelators and/or may include compounds orcompositions otherwise capable of decreasing the copper values contentof the patient (for example; zinc (e.g.; as a suitable salt such as thegluconate) or trithiomolybdate (also a copper chelator) which tend toprevent copper absorption by a patient).

The method may include an additional step or steps of evaluating ormonitoring the copper values of the patient prior to, simultaneouslywith and/or subsequent to the patient being subjected to or beingadministered with the agent(s). The method may also include, forexample, diagnosis of the patient as a diabetic.

In still another aspect the present invention provides a method oftreating a mammal (for example, a human being) at risk of developing,with suspected or with actual diabetic hypertensive cardiomyopathy whichcomprises or includes the step of subjecting the patient mammal toand/or administering to the patient one or more agents capable ofdecreasing the copper values content of the patient. Such agent(s) maycomprise or include copper chelators and/or may include compounds orcompositions otherwise capable of decreasing the copper values contentof the patient (for example, zinc (for example, as a suitable salt suchas the gluconate form) or trithiomolybdate (also a copper chelator)which tend to prevent copper absorption by a patient). The method mayinclude an additional step or steps of evaluating or monitoring thecopper values of the patient prior to, simultaneously with and/orsubsequent to the patient being subjected to or being administered withthe agent(s). The method may also include, for example, diagnosis of thepatient as a diabetic.

In yet another aspect the present invention provides a method oftreating a mammal (for example, a human being) at risk of developing,with suspected or with actual acute myocardial infarction (AMI)associated with impaired glucose tolerance (IGT) which comprises orincludes the step of subjecting the patient mammal to and/oradministering to the patient one or more agents capable of reducing thecopper values content of the patient. Such agent(s) may comprise orinclude copper chelators and/or may include compounds or compositionsotherwise capable of reducing the copper values content of the patient(for example, zinc (for example, as a suitable salt including thegluconate form) or trithiomolybdate (also a copper chelator) which tendto prevent copper absorption by a patient). The method may include anadditional step or steps of evaluating or monitoring the copper valuesof the patient prior to, simultaneously with and/or subsequent to thepatient being subjected to or being administered with the agent(s). Themethod may also include diagnosis of the patient, for example, as adiabetic. The method may also include one or both of the additionalsteps of diagnosis of the patient with myocardial infarction and/orimpaired glucose tolerance.

In another aspect the present invention provides a method of treating amammal (for example, a human being) at risk of developing, withsuspected or with actual acute myocardial infarction associated withimpaired fasting glucose (IFG) which comprises or includes the step ofsubjecting the patient mammal to and/or administering to the patientmammal one or more agents capable of reducing the copper values contentof the patient. Such agent(s) may comprise or include copper chelatorsand/or may include compounds or compositions otherwise capable ofreducing the copper values content of the patient (for example, zinc(for example, as a suitable salt including the gluconate form) ortrithiomolybdate (also a copper chelator) which tend to prevent copperabsorption by a patient). The method may include an additional step orsteps of evaluating or monitoring the copper values of the patient priorto, simultaneously with and/or subsequent to the patient being subjectedto or being administered with the agent(s). The method may also includediagnosis of the patient, for example, as a diabetic. The method canalso include one or both of the additional steps of diagnosis of thepatient with myocardial infarction and/or impaired fasting glucose.

In still another aspect the present invention provides a method oftreating a mammal (for example, a human being) at risk of developing,with suspected or with actual hypertensive cardiomyopathy associatedwith IGT which comprises or includes the step of subjecting the patientmammal to and/or administering to the patient mammal one or more agentscapable of reducing the copper values content of the patient. Suchagent(s) may comprise or include copper chelators and/or may includecompounds or compositions otherwise capable of reducing the coppervalues content of the patient (for example, zinc (for example, as asuitable salt such as the gluconate form) or trithiomolybdate (also acopper chelator) which tend to prevent copper absorption by a patient).The method may also include an additional step or steps of evaluating ormonitoring the copper values of the patient prior to, simultaneouslywith and/or subsequent to the patient being subjected to or beingadministered with the agent(s). The method may also include diagnosis ofthe patient, for example, as a diabetic, The method may also include theadditional step or steps of diagnosing the patient as a hypertensiveand/or as being subjected to IGT and/or suffering from actualhypertensive cardiomyopathy.

In yet another aspect the present invention provides a method oftreating a mammal (for example, a human being) at risk of developing,with suspected or with actual hypertensive cardiomyopathy associatedwith IFG which comprises or includes the step of subjecting the patientmammal to and/or administering to the patient mammal one or more agentscapable of reducing the copper values content of the patient Suchagent(s) may comprise or include copper chelators and/or may includecompounds or compositions otherwise capable of reducing the coppervalues content of the patient (for example; zinc (for example, as asuitable salt such as the gluconate) or trithiomolybdate (also a copperchelator) which tend to prevent copper absorption by a patient). Themethod may also include an additional step or steps of evaluating ormonitoring the copper values of the patient prior to, simultaneouslywith and/or subsequent to the patient being subjected to or beingadministered with the agent(s). The method may also include diagnosis ofthe patient, for example, as a diabetic. The method may further includethe additional step or steps of diagnosing the patient, for example, asa hypertensive and/or having IFG and/or having hypertensivecardiomyopathy.

In another aspect the present invention provides a method of treating amammal (for example, a human being) at risk of developing, withsuspected or with actual ischaemic cardiomyopathy associated with IGTwhich comprises or includes the step of subjecting the patient mammal toand/or administering to the patient mammal one or more agents capable ofreducing the copper values content of the patient. Such agent(s) maycomprise or include copper chelators and/or may include compounds orcompositions otherwise capable of decreasing the copper values contentof the patient (for example, zinc (for example, as a suitable salt suchas the gluconate form) or trithiomolybdate (also a copper chelator)which tend to prevent copper absorption by a patient). The method mayalso include an additional step or steps of evaluating or monitoring thecopper values of the patient prior to, simultaneously with and/orsubsequent to the patient being subjected to or being administered withthe agent(s). The method may further include diagnosis of the patient,for example, as a diabetic. The method may also include the additionalstep of determining the patient is subject to ischemic disease and/or issubject to IGT and/or is suffering from ischemic cardiomyopathy.

In yet another aspect the present invention provides a method oftreating a mammal (for example, a human being) at risk of developing,with suspected or with actual ischaemic cardiomyopathy associated withIFG which comprises or includes the step of subjecting the patientmammal to and/or administering to the patient mammal one or more agentscapable of decreasing the copper values content of the patient. Suchagent(s) may comprise or include copper chelators and/or may includecompounds or compositions otherwise capable of decreasing the coppervalues content of the patient (for example, zinc (for example, as asuitable salt such as the gluconate form) or trithiomolybdate (also acopper chelator) which tend to prevent copper absorption by a patient).The method may include an additional step or steps of evaluating ormonitoring the copper values of the patient prior to, simultaneouslywith and/or subsequent to the patient being subjected to or beingadministered with the agent(s). The method may also include diagnosis ofthe patient, for example, as a diabetic. The method may include theadditional step or steps of diagnosing the patient as ischaemic and/orhaving IFG and/or suffering from ischaemic cardiomyopathy. The methodmay include the additional step or steps of diagnosing the patient assubject to ischaemic disease and/or suffering from coronary heartdisease (CHD) and/or suffering from ischaemic cardiomyopathy.

In another aspect the present invention provides a method of treating amammal (for example, a human being) at risk of developing, withsuspected or with actual ischaemic cardiomyopathy associated withcoronary heart disease (CHD) which comprises or includes the step ofsubjecting the patient mammal to and/or administering to the patientmammal one or more agents capable of decreasing the copper valuescontent of the patient. Such agent(s) may comprise or include copperchelators and/or may include compounds or compositions otherwise capableof decreasing the copper values content of the patient (for example,zinc (for example, as a suitable salt such as the gluconate from) ortrithiomolybdate (also a copper chelator) which tend to prevent copperabsorption by a patient). The method may include an additional step orsteps of evaluating or monitoring the copper values of the patient priorto, simultaneously with and/or subsequent to the patient being subjectedto or being administered with the agent(s). The method may also includediagnosis of the patient, for example, as a diabetic. The method mayinclude the additional step or steps of diagnosing the patient assuffering from acute myocardial infarction.

In another aspect the present invention provides a method of treating amammal (for example, a human being) at risk of developing, withsuspected or with actual acute myocardial infarction not associated withany abnormality of the glucose metabolism which comprises or includesthe step of subjecting the patient mammal to and/or administering to thepatient mammal one or more agents capable of decreasing the coppervalues content of the patient. Such agent(s) may comprise or includecopper chelators and/or may include compounds or compositions otherwisecapable of decreasing the copper values content of the patient (forexample, zinc (for example, as a suitable salt such as the gluconate) ortrithiomolybdate (also a copper chelator) which tend to prevent copperabsorption by a patient). The method may include an additional step orsteps of evaluating or monitoring the copper values of the patient priorto, simultaneously with and/or subsequent to the patient being subjectedto or being administered with the agent(s). The method may include theadditional step or steps of diagnosing the patient, for example, ashypertensive and/or suffering from hypertensive cardiomyopathy.

In another aspect the present invention provides a method of treating amammal (for example, a human being) at risk of developing, withsuspected or with actual hypertensive cardiomyopathy not associated withany abnormality of the glucose metabolism which comprises or includesthe step of subjecting the patient mammal to and/or administering to thepatient one or more agents capable of decreasing the copper valuescontent of the patient. Such agent(s) may comprise or include copperchelators and/or may include compounds or compositions otherwise capableof decreasing the copper values content of the patient (for example,zinc (for example, as a suitable salt such as the gluconate form) ortrithiomolybdate (also a copper chelator) which tend to prevent copperabsorption by a patient). The method may include an additional step orsteps of evaluating or monitoring the copper values of the patient priorto, simultaneously with and/or subsequent to the patient being subjectedto or being administered with the agent(s). The method may also includediagnosis of the patient, for example, as a diabetic. The method mayalso include the additional step or steps of diagnosing the patient, forexample, as hypertensive and/or suffering from hypertensivecardiomyopathy.

In yet a further aspect the present invention provides a method oftreating a mammal (for example, a human being) at risk of developing,with suspected or with actual ischemic cardiomyopathy not associatedwith any abnormality of the glucose metabolism (irrespective of whetheror not such ischemic cardiomyopathy is associated with coronary heartdisease or not) which comprises or includes the step of subjecting thepatient mammal to and/or administering to the patient one or more agentscapable of decreasing the copper values content of the patient. Suchagent(s) may comprise or include copper chelators and/or may includecompounds or compositions otherwise capable of decreasing the coppervalues content of the patient (for example, zinc (for example, as asuitable salt such as the gluconate form) or trithiomolybdate (also acopper chelator) which tend to prevent copper absorption by a patient).The method may include an additional step or steps of evaluating ormonitoring the copper values of the patient prior to, simultaneouslywith and/or subsequent to the patient being subjected to or beingadministered with the agent(s). The method may also include diagnosis ofthe patient, for example, as a diabetic. The method may include theadditional step or steps of diagnosing the patient as suffering from,for example, ischemic disease and/or ischemic cardiomyopathy.

In a further aspect the present invention provides a method of treatinga human at risk of developing, with suspected or with actualcardiomyopathy which comprises or includes the steps of categorizing thehuman by reference to (a) whether suffering from one or more of type 2diabetes, impaired glucose tolerance (IGT) and impaired fasting glucose(IFG), and/or (b) copper status, and (provided the patient (a) issuffering from type 2 diabetes and/or (IGT) and/or IFG, and/or (b) isnot biochemically or clinically or undesirably copper deficient)subjecting the patient to a regimen with a view to decreasing thepresence of copper values. There may also be a step of ensuring byreference to heart function that the patient is benefiting from thecopper decreasing regimen.

In a further aspect the present invention provides a method of treatinga human at risk to developing, with suspected or with actual acutemyocardial infarction which comprises or includes the steps ofcategorizing the human by reference to (a) whether suffering from one ormore Type 2 diabetes, impaired glucose tolerance (IGT) and impairedfasting glucose (IFG), and/or (b) copper status, and (provided thepatient (a) is suffering from Type 2 diabetes and/or IGT and/or IGF,and/or (b) is not biochemically or clinically or undesirably copperdeficient) subjecting the patient to an copper chelation and/or othercopper values decreasing regimen with a view to decreasing the presenceof copper. Step (i) may also includes reference to (c) heart function.Alternatively and/or additionally benefit to patient is assessed byreference to heart function.

In another aspect the present invention provides a method of treating ahuman at risk of developing, with suspected or with actual hypertensivecardiomyopathy which comprises or includes the steps of categorizing thehuman by reference to (a) whether hypertensive, and/or (b) copperstatus; and subjecting the patient to an copper chelation and/or othercopper values decreasing regimen with a view to decreasing the presenceof copper whilst preferably ensuring patient does not have or does notdevelop a copper deficiency. Step (i) may also include one or bothreferences to (b) copper status and/or (c) heart function. There mayalso be a step of ensuring by reference to heart function that thepatient is benefiting from the copper chelation regimen.

In a further aspect the present invention provides a method of treatinga human at risk to developing, with suspected or with actual ischemiccardiomyopathy which comprises or includes the steps of categorizing thehuman by reference to (a) whether suffering from ischaemia, and/or (b)copper status; and subjecting the patient to a copper chelation and/orother copper values decreasing regimen with a view to decreasing thepresence of copper whilst preferably ensuring patient does not have ordoes not develop any copper deficiency.

In still another aspect the present invention provides a method oftreating a human at risk of developing, with suspected or with actualcardiomyopathy which comprises or includes the steps of categorizing thehuman as a candidate patient by reference to at least (a) whethersuffering from Type 2 diabetes, (IGT), impaired fasting glucose (IFG)and/or hypertensive impaired glucose tolerance, and (b) heart function,and subjecting the patient to an copper chelation and/or other coppervalues decreasing regimen with a view to decreasing the presence ofcopper whilst preferably ensuring patient does not have or does notdevelop any copper deficiency. There may also be a step of ensuring byreference to heart function that the patient is benefiting from thecopper chelation regimen.

In a further aspect the present invention provides a method of treatinga human or other mammal at risk to developing, with suspected or withactual (I) arterial, (II) arterial and coronary and/or other organ,and/or (III) heart muscle disease which comprises or includes the stepsof categorizing the human or other mammal as a candidate patient andsubjecting the patient to an copper chelation and/or other copper valuesdecreasing regimen with a view to decreasing the presence of copper.Step (i) may also include a determination of the copper status of thehuman or other mammal. There may also be a step of ensuring by referenceto heart and/or arterial function that the patient is benefiting fromthe copper chelation and/or other copper values decreasing regimen.

In another aspect, the one or more agents capable of decreasing thecopper values content of the patient, if a chelator, has a preferentialaffinity for copper values over the values of other trace metals (suchas iron, zinc and/or manganese). Preferably the preferential affinityfor copper values is such that copper excess of from about 100% to about500% over that of a normal healthy mammal of the species can becontrolled to such normal levels or approaching such normal levelswithout leading to depletion or excessive decreases in iron, zinc and/ormanganese. With the chelators such as those herein referred to and thesuitable salts of zinc it is possible to selectively decrease the coppervalues in the body as a whole even though it is believed there is littledecrease in copper values in the intra cellular tissue. Without beingbound by this mechanism, it is believed that the decrease is primarilyextracellular (for example, interstitial, on the exterior of cellsand/or on collagen).

In another aspect the present invention provides a method of treatment,for example, that includes the methodology of either FIG. 3 or 4 of theaccompanying drawings.

In a further aspect the present invention provides a method of treatinga human having type 2 diabetes or impaired glucose intolerance at riskof developing, with suspected or with actual cardiomyopathy whichcomprises or includes subjecting the patient to an copper chelationregimen with a view to decreasing the presence of chelatable copper toheart tissue whilst at least on occasions having monitored and/orevaluating or monitoring the patient to avoid a copper deficit. Thepatient may also be or have been categorized to ensure that the regimenis not commenced and/or does not continue should the patient be copperdeficient.

In a further aspect the present invention provides a method of treatinga human having type 2 diabetes or impaired glucose intolerance at riskof developing, with suspected or with actual macrovascular disease whichcomprises or includes subjecting the patient to a total body coppercontent decreasing regimen. The patient may also be or have beencategorized to ensure that the regimen is not commenced and/or does notcontinue should the patient be copper deficient.

In still a further aspect or as one preferment the present inventionprovides a method of treating a human at risk of developing, withsuspected or with actual cardiomyopathy related heart failure whichcomprises or includes decreasing the levels of chelatable and/or othercopper values of such patient preferably without taking the patient intoundesirable copper values or copper deficit.

In yet a further aspect the present invention provides a method oftreating a human at risk of developing, with suspected or with actualmacrovascular disease of the arterial tree which comprises or includesdecreasing the levels of chelatable copper in the walls of major bloodvessels of such patient without taking the patient into undesirablecopper values or copper deficit.

In yet another aspect the present invention provides a method oftreating a human at risk of developing, with suspected or with actualcardiomyopathy which comprises or includes the steps of categorizing thehuman as being at risk of developing, with suspected or with actualcardiomyopathy, and subjecting the patient to an copper chelationregimen with a view to decreasing the presence of copper. The copperchelation regimen may include or be subject to evaluating or monitoringto ensure the patient does not have or does not develop undesirablecopper values or a copper deficiency. The categorization may rely on aninitial check of heart function and the patient being categorized forthe copper chelation regimen when that heart function is below normal.The heart function evaluating or monitoring may also continue into orbeyond the copper chelation regimen. The categorization may include adetermination of the patient suffering from type 2 diabetes or impairedglucose tolerance. The categorization may also involve a reference tocopper status of the patient prior to any commencement or substantialduration of the copper chelation regimen to ensure the patient does nohave or does not develop undesirable copper values or a copperdeficiency.

In any of the foregoing procedures the following (any one, some or all)arise or be involved. The compound may be a copper chelator which in themammal is substantially without an ability to generate free radicals insignificant qualities and which also in the mammal at the dosage regimento be given will not chelate copper down to a depletion state in themammal. The administration is at a dosage regimen less than that whichfor a patient suffering from classical copper overload would have theeffect of decreasing the copper levels of that patient to normal. Theadministration is at a dosage regimen (whether dependent upon dosageunit(s) and/or frequency) that does not or will not reduce a patient ofnormal copper levels to a deficiency state. The regimen is in concert(serial, simultaneous or otherwise) with a regimen to antagonizefructosamine oxidase. The dosage unit(s) is (are) the dosage unit(s) ofa copper decreasing regimen. The regimen may run in concert with any ofthe regimens disclosed in WO 00/18392. The use involves pharmaceuticallyacceptable diluents and/or carriers. The composition is for use in amethod referenced, suggested or identified herein.

The present invention also provides a dosage unit resulting from or forany such use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing various pathways addressed by the presentinvention.

FIG. 2 is a hypothesis of the mechanisms involved applicable tocardiomyopathy and macrovascular disease in a patient with type 2diabetes or impaired glucose tolerance, for example, such a hypothesisshowing reliance on a possible fructosamine oxidase/superoxide dismutasegeneration of a precursor to an copper catalyzed reaction (theHaber-Weiss Reaction) which generates the harmful free radicals.

FIG. 3 is the methodology for a human patient with suspectedcardiomyopathy under the present invention.

FIG. 4 is a similar diagram to that of FIG. 3 but in respect of apatient with suspected macrovascular disease.

FIG. 5 is a diagram showing the body weight of animals changing over thetime period of experiment.

FIG. 6 shows the glucose levels of the animals changing over the timeperiod of the experiment.

FIG. 7 is a diagram showing cardiac output.

FIG. 8 is a diagram showing coronary flow.

FIG. 9 is a diagram showing coronary flow normalized to final cardiacweight.

FIG. 10 is a diagram showing aortic flow.

FIG. 11 is a diagram showing the maximum rate of positive change inpressure development in the ventricle with each cardiac cycle(contraction).

FIG. 12 is a diagram showing the maximum rate of decrease in pressure inthe ventricle with each cardiac cycle (relaxation).

FIG. 13 shows the percentage of functional surviving hearts at eachafter-load.

FIG. 14 shows diagrammatically how the extracted heart was attached tothe modified apparatus.

FIG. 15 shows diagrammatically the heart depicted in FIG. 14 in moredetail (picture adapted from Grupp I et al., Am J Physiol 34:111401-1410(1993)).

FIG. 16 shows the urine excretion in diabetic and non diabetic animalsin response to increasing doses of trientine or equivalent volume ofsaline, wherein urine excretion in diabetic and nondiabetic animals inresponse to increasing doses of trientine (bottom; 0.1, 1.0, 10, 100mg·kg⁻¹ in 75 μl saline followed by 125 μl saline flush injected at timeshown by arrow) or an equivalent volume of saline (top), and each pointrepresents a 15 min urine collection period (see Methods for details);error bars show SEM and P values are stated if significant (P<0.05).

FIG. 17 shows urine excretion in non diabetic and diabetic animalsreceiving increasing doses of trientine or an equivalent volume ofsaline, wherein urine excretion in diabetic (top) and nondiabetic(bottom) rats receiving increasing doses of trientine (0.1, 1.0, 10, 100mg·kg⁻¹ in 75 μA saline followed by 125 μl saline flush injected at timeshown by arrow) or an equivalent volume of saline, and each pointrepresents a 15 ruin urine collection period (see Methods for details);error bars show SEM and P values are stated if significant (P<0.05).

FIG. 18 shows copper excretion in the urine of diabetic and non diabeticanimals receiving increasing doses of trientine or an equivalent volumeof saline, wherein copper excretion in urine of diabetic (top) andnondiabetic (bottom) rats receiving increasing doses of trientine (0.1,1.0, 10, 100 g·kg⁻¹ in 75 μl saline followed by 125 μl saline flushinjected at time shown by arrow) or an equivalent volume of saline, andeach point represents a 15 min urine collection period (see Methods fordetails); error bars show SEM and P values are stated if significant(P<0.05).

FIG. 19 shows the same information in FIG. 18 with presentation ofurinary copper excretion per gram of bodyweight, wherein urinary copperexcretion per gram of bodyweight in diabetic and nondiabetic animals inresponse to increasing doses of trientine (bottom; 0.1, 1.0, 10, 100mg·kg⁻¹ in 75 μl saline followed by 125 μl saline flush injected at timeshown by arrow) or an equivalent volume of saline (top), and each pointrepresents a 15 min urine collection period (see Methods for details);error bars show SEM and P values are stated if significant (P<0.05).

FIG. 20 shows the total amount of copper excreted in non diabetic anddiabetic animals administered saline or drug, wherein total urinarycopper excretion (mmol) in nondiabetic animals administered saline(black bar, n=7) or trientine (hatched bar, n=7) and in diabetic animalsadministered saline (grey bar, n=7) or trientine (white bar, n=7); errorbars show SEM and P values are stated if significant (P<0.05).

FIG. 21 shows the total amount of copper excreted per gram of bodyweightin animals receiving trientine or saline, wherein total urinary copperexcretion per gram of bodyweight (μgμgBW⁻¹) in animals receivingtrientine (nondiabetic: hatched bar, n=7; diabetic: white bar, n=7) orsaline (nondiabetic: black bar, n=7; diabetic: grey bar, n=7); errorbars show SEM and P values are stated if significant (P<0.05).

FIG. 22 shows the iron excretion in urine of diabetic and non diabeticanimals receiving increasing doses of trientine or an equivalent volumeof saline, wherein iron excretion in urine of diabetic (top) andnondiabetic (bottom) rats receiving increasing doses of trientine (0.1,1.0, 10, 100 mg·kg⁻¹ in 75 μl saline followed by 125 μl saline flushinjected at time shown by arrow) or an equivalent volume of saline, andeach point represents a 15 min urine collection period (see Methods fordetails); error bars show SEM and P values are stated if significant(P<0.05).

FIG. 23 shows the urinary iron excretion per gram of bodyweight indiabetic and non diabetic animals receiving trientine or saline, whereinurinary iron excretion per gram of bodyweight in diabetic andnondiabetic animals in response to increasing doses of trientine(bottom; 0.1, 1.0, 10, 100 mg·kg⁻¹ in 75 μl saline followed by 125 μlsaline flush injected at time shown by arrow) or an equivalent volume ofsaline (top), and each point represents a 15 min urine collection period(see Methods for details); error bars show SEM and P values are statedif significant (P<0.05).

FIG. 24 shows the total urinary iron excretion in non diabetic anddiabetic animals administered saline or drug, wherein total urinary ironexcretion (μmol) in nondiabetic animals administered saline (black bar,n=7) or trientine (hatched bar, n=7) and in diabetic animalsadministered saline (grey bar, n=7) or trientine (white bar, n=7); errorbars show SEM and P values are stated if significant (P<0.05).

FIG. 25 shows the total urinary iron excretion per gram of bodyweight inanimals receiving trientine or saline, wherein Total urinary ironexcretion per gram of bodyweight (μg·gBW⁻¹) in animals receivingtrientine (nondiabetic: hatched bar, n=7; diabetic: white bar, n=7) orsaline (nondiabetic: black bar, n=7; diabetic: grey bar, n=7); errorbars show SEM and P values are stated if significant (P≦0.05).

FIG. 26 shows the percentage of surviving hearts at each after-loadpressure.

FIG. 27 is a table comparing the copper and iron excretion in theanimals receiving trientine or saline, which is a statistical analysisusing a mixed linear model.

DETAILED DESCRIPTION OF THE INVENTION

The invention is related to and describes the methods relating todiscoveries surrounding increased tissue copper and mechanisms leadingto tissue damage, including nerve and vascular damage, for example,diabetic nerve and/or vascular damage. It is believed, without wishingto be bound by any particular mechanism or theory of operation oreffectiveness, that tissue accumulation of trace metals plays a role inthe mechanisms of tissue damage in diabetes as well as in otherdisorders, diseases, and conditions as set forth or referenced orsuggested herein.

Histological evidence from experiments showed that six months oftreatment with trientine appears to protect the hearts of diabeticWistar rats from development of diabetic damage (cardiomyopathy) asjudged by histology. The doses of trientine required for copper and ironto be excreted in the urine have also been investigated, for example, aswell as possible differences between the excretion of these metals indiabetic and nondiabetic animals. For example, the excretion profiles ofcopper and iron in the urine of normal and diabetic rats were comparedafter acute intravenous administration of increasing doses of trientine.Additionally, it was ascertained whether acute intravenousadministration of trientine has acute adverse cardiovascular sideeffects. Methods used in the experimentals were as follows.

Male Wistar rats (n=28, 303±2.9 g) were divided randomly into diabeticand nondiabetic groups. Following induction of anesthesia (5% halothaneand 21.min⁻¹ O₂), animals in the diabetic group received a singleintravenous dose of streptozotocin (STZ, 55 mg·kg⁻¹ body weight, Sigma;St. Louis, Mo.) in 0.5 ml saline administered via the tail vein.Nondiabetic animals received an equivalent volume of saline. Followinginjection, both diabetic and nondiabetic rats were housed in like-pairsand provided with access to normal rat chow (Diet 86 pellets; NewZealand Stock Feeds, Auckland, NZ) and deionized water ad libitum. Bloodglucose and body weight were measure at day 3 following STZ/salineinjection and then weekly throughout the study. Diabetes was identifiedby polydipsia, polyuria and hyperglycemia (>11 mmol·l⁻¹, Advantage II,Roche Diagnostics, NZ Ltd).

Six to seven weeks (mean=44±1 days) after administration of STZ, animalsunderwent either a control or drug experimental protocol. All animalswere fasted overnight prior to surgery but continued to have ad libitumaccess to deionized water. Induction and maintenance of surgicalanesthesia was by 3-5% halothane and 21.min⁻¹ O₂. The femoral artery andvein were cannulated with a solid-state blood pressure transducer(Mikratip™ 1.4 F, Millar Instruments, Texas, USA) and a saline filled PE50 catheter respectively. The ureters were exposed via a midlineabdominal incision, cannulated using polyethylene catheters (externaldiameter 0.9 mm, internal diameter 0.5 mm) and the wound sutured closed.The trachea was cannulated and the animal ventilated at 70-80breaths·min⁻¹ with air supplemented with O₂ (Pressure ControlledVentilator, Kent Scientific, Connecticut, USA). The respiratory rate andend-tidal pressure (10-15 cmH₂O) were adjusted to maintain end-tidal CO₂at 35-40 mm Hg (SC-300 CO₂ Monitor, Pryon Corporation, Wisconsin, USA).Body temperature was maintained at 37° C. throughout surgery and theexperiment by a heating pad. Estimated fluid loss was replaced withintravenous administration of 154 mmol·l⁻¹ NaCl solution at a rate of 5ml·kg⁻¹·h⁻¹.

Following surgery and a 20 min stabilization period, the experimentalprotocol was started. Trientine was administered intravenously over 60 sin hourly doses of increasing concentration (0.1, 1.0, 10 and 100mg·kg⁻¹ in 75 μl saline followed by 125 μl saline flush). Controlanimals received an equivalent volume of saline. Urine was collected in15 min aliquots throughout the experiment in pre-weighed polyethyleneepindorph tubes. At the end of the experiment a terminal blood samplewas taken by cardiac puncture and the separated serum stored at −80° C.until future analysis. Hearts were removed through a rapid mid-sternalthoracotomy and processed as described below.

Mean arterial pressure (MAP), heart rate (HR, derived from the MAPwaveform) oxygen saturation (Nonin 8600V Pulse Oximeter, Nonin MedicalInc., Minnesota, USA) and core body temperature, were all continuouslymonitored throughout the experiment using a PowerLab/16s dataacquisition module (AD Instruments, Australia). Calibrated signals weredisplayed on screen and saved to disc as 2 s averages of each variable.

Instrumentation: A Perkin Elmer (PE) Model 3100 Atomic AbsorptionSpectrophotometer equipped with a PE HGA-600 Graphite Furnace and PEAS-60 Furnace Autosampler was used for Cu and Fe determinations inurine. Deuterium background correction was employed. A Cu or Fehollow-cathode lamp (Perkin Elmer Corporation) was used and operated ateither 10 W (Cu) or 15 W (Fe). The 324.8 nm atomic line was used for Cuand the 248.3 nm atomic line for Fe. The slit width for both Cu and Fewas 0.7 nm. Pyrolytically coated graphite tubes were used for allanalyses. The injection volume was 20 μL. A typical graphite furnacetemperature program is shown below.

GF-AAS temperature program Procedure Temp/° C. Ramp/s Hold/s Int.Flow/mL min⁻¹ Drying  90 1 5 300  120 60 5 300 Pre-treatment 1250* 20 10300  20 1 10 300 Atomization - Cu/Fe 2300/2500 1 8 0 Post-treatment 26001 5 300 *A pre-treatment temperature of 1050° C. was used for tissuedigest analyses

Cu, Fe and Zn in tissue digests were also determined at HillLaboratories (Hamilton, New Zealand) using either a PE Sciex Elan-6000or PE Sciex Elan-6100 DRC ICP-MS. The operating parameters aresummarized in the table below.

Instrumental operating parameters for ICP-MS Parameter Value Inductivelycoupled plasma Radiofrequency power 1500 W Argon plasma gas flow rate 15l · min⁻¹ Argon auxiliary gas flow rate 1.2 l · min⁻¹ Argon nebulisergas flow rate 0.89 l · min⁻¹ Interface Sampler cone and orifice diameterNi/1.1 mm Skimmer cone and orifice diameter Ni/0.9 mm Data acquisitionparameters Scanning mode Peak hopping Dwell time 30 ms (Cu, Zn)/100 ms(Fe) Sweeps/replicate 20 Replicates  3 Sample uptake rate 1 ml · min⁻¹

Reagents: All reagents used were of the highest purity available and atleast of analytical grade. GF-AAS standard working solutions of Cu andFe were prepared by stepwise dilution of 1000 mg·l⁻¹ (Spectrosolstandard solutions; BDH). Water was purified by a Millipore Milli-Qultra-pure water system to a resistivity of 18 Ma Standard ReferenceMaterial 1577b Bovine Liver was obtained from the National Institute ofStandards and Technology and used to evaluate the efficiency of tissuedigestion. The results obtained are reported below.

GF-AAS and ICP-MS results for NIST SRM 1577b bovine liver* ElementCertified value GF-AAS ICP-MS Cu 160 ± 8  142 ± 12 164 ± 12 Fe 184 ± 15182 ± 21 166 ± 14 Zn 127 ± 16 — 155 ± 42 *Measured in μg · g⁻¹ of drymatter.

Samples were pretreated as follows:

Urine:

Urine was collected in pre-weighed 1.5 ml micro test tubes (eppendorf).After reweighing, the urine specimens were centrifuged and thesupernatant diluted 25:1 with 0.02 M 69% Aristar grade HNO₃. The samplewas stored at 4° C. prior to GF-AAS analysis. If it was necessary tostore a sample for a period in excess of 2 weeks, it was frozen and keptat −20° C.

Heart:

Following removal from the animal, the heart was cleaned of excesstissue, rinsed in buffer to remove excess blood, blotted dry and a wetventricular weight recorded. Using titanium instruments a segment ofleft ventricular muscle was dissected and placed in a pre-weighed 5.0 mlpolystyrene tube. The sample was freeze-dried overnight to constantweight before 0.45 ml of 69% Aristar grade HNO₃ was added. The sampletube was heated in a water bath at 65° C. for 60 minutes. The sample wasbrought to 4.5 ml with Milli-Q H₂O. The resulting solution was diluted2:1 in order to reduce the HNO₃ concentration below the maximumpermitted for ICP-MS analysis.

Serum:

Terminal blood samples were centrifuged and serum treated and stored asper urine until analysis. From the trace metal content of serum from theterminal blood sample and urine collected over the final hour of theexperiment, renal clearance was calculated using the following equation:renal clearance of trace metal=(a) the concentration of metal in urine(μg·μl⁻¹) times (b) the rate of urine flow (μl·min⁻¹), divided by (c)the concentration of metal in serum (μg·μl⁻¹)

Statistical analyses were as follows: All values are expressed asmean±SEM and P values<0.05 were considered statistically significant.Student's unpaired t-test was initially used to test for weight andglucose differences between the diabetic and control groups. Forcomparison of responses during drug exposure, statistical analyses wereperformed using analysis of variance (Statistica for Windows v.6.1, SASInstitute Inc., California, USA). Subsequent statistical analysis wasperformed using a mixed model repeated measures ANOVA design.

Statistical analysis using a mixed linear model: Data for each doselevel were analyzed using a mixed linear model (PROC MIXED; SAS, Version8). The model included diabetes, drug and their interaction as fixedeffects, time as a repeated measure, and rats as the subjects in thedataset. Complete independence is assumed across subjects. The fullmodel was fitted to each dataset using a maximum likelihood estimationmethod (REML) fits mixed linear models (i.e., fixed and random effectsmodels). A mixed model is a generalization of the standard linear model,the generalization being that you can analyse data generated fromseveral sources of variation instead of just one. A level ofsignificance of 0.05 was used for all tests. The results were asfollows.

Effects of STZ on blood glucose and body weight (Table 1): Blood glucoseincreased to 25±2 mmol·l⁻¹ three days following STZ injection. Despite agreater daily food intake, diabetic animals lost weight whilstnondiabetic animals continued to gain weight during the 44 daysfollowing STZ/saline injection. On the day of the experiment bloodglucose levels were 24±1 and 5±0 mmol·l⁻¹ and body weight 264±7 g and434±9 g for diabetic and nondiabetic animals respectively.

TABLE 1 Blood glucose, body weight and food consumption in diabeticversus nondiabetic animals. I. DIABETIC II. NONDIABETIC Body weightprior to  303 ± 3 g 303 ± 3 g STZ/saline Blood glucose 3 days  *25 ± 2mmol · l⁻¹  5 ± 0.2 mmol · l⁻¹ following STZ/saline Daily foodconsumption  *58 ± 1 g  28 ± 1 g Blood glucose on  *24 ± 1 mmol · l⁻¹  5± 0.2 mmol · l⁻¹ experimental day Body weight on *264 ± 7 g 434 ± 9 gexperimental day Diabetic animals n = 14, nondiabetic animals n = 14.Values shown as mean ± SEM. Asterisk indicates a significant difference(P < 0.05).

Cardiovascular variables during infusion: Baseline levels of MAP duringthe control period prior to infusion were not significantly differentbetween nondiabetic and diabetic animals (99±4 mm Hg). HR wassignificantly lower in diabetic than nondiabetic animals (287±11 and364±9 bpm respectively, P<0.001). Infusion of trientine or saline had noeffect on these variables except at the highest dose where MAP decreasedby a maximum of 19±4 mm Hg for the 2 min following administration andreturned to pre-dose levels within 10 min. Body temperature and oxygensaturation remained stable in all animals throughout the experiment.

Urine excretion: Diabetic animals consistently excreted significantlymore urine than nondiabetic animals except in response to the highestdose of drug (100 mg·kg⁻¹) or equivalent volume of saline (FIG. 16).Administration of the 100 mg·kg⁻¹ dose of trientine also increased urineexcretion in nondiabetic animals to greater than that of nondiabeticanimals receiving the equivalent volume of saline (FIG. 17). This effectwas not seen in diabetic animals.

Urinary excretion of Cu and Fe: Analysis of the dose response curvesshows that, at all doses, diabetic and nondiabetic animals receivingdrug excreted more Cu than animals receiving an equivalent volume ofsaline (FIG. 18). To provide some correction for the effects of lessertotal body growth of the diabetic animals, and thus to allow moreappropriate comparison between diabetic and nondiabetic animals,excretion rates of trace elements were also calculated per gram of bodyweight. FIG. 19 shows that diabetic animals had significantly greatercopper excretion per gram of body weight in response to each dose ofdrug than did nondiabetic animals. The same pattern was seen in responseto saline, however the effect was not always significant. Total copperexcreted over the entire duration of the experiment was significantlyincreased in both nondiabetic and diabetic animals administeredtrientine compared with their respective saline controls (FIG. 20).Diabetic animals receiving drug also excreted more total copper per gramof body weight than nondiabetic animals receiving drug. The samesignificant trend was seen in response to saline administration (FIG.21).

In comparison, iron excretion in both diabetic and nondiabetic animalsreceiving trientine was not greater than animals receiving an equivalentvolume of saline (FIG. 22). Analysis per gram of body weight showsdiabetic animals receiving saline excrete significantly more iron thannondiabetic animals, however this trend was not evident between diabeticand nondiabetic animals receiving trientine (FIG. 23). Total ironexcretion in both diabetic and nondiabetic animals receiving drug wasnot different from animals receiving saline (FIG. 24). In agreement withanalysis of dose response curves, total iron excretion per gram of bodyweight was significantly greater in diabetic animals receiving salinethan nondiabetic animals but this difference was not seen in response totrientine (FIG. 25).

Serum content and renal clearance of Cu and Fe (Table 2): While therewas no significant difference in serum copper content, there was asignificant increase in renal clearance of copper in diabetic animalsreceiving drug compared with diabetic animals receiving saline. The samepattern was seen in nondiabetic animals, although the trend was notstatistically significant (P=0.056). There was no effect of drug orstate (diabetic versus nondiabetic) on serum content or renal clearanceof iron.

TABLE 2 Serum content and renal clearance of Cu and Fe in diabetic andnondiabetic animals receiving drug or saline. 1.1.a.a.1 1.1.a.a.11.1.a.a.2 1.1.a.a.2 diabetic diabetic nondiabetic nondiabetic trientineSaline trientine Saline n = 6 n = 7 n = 4 n = 7 Serum Cu 7.56 ± 0.069.07 ± 1.74 7.11 ± 0.41 7.56 ± 0.62 (μg · μl⁻¹ × 10⁻⁴) Serum Fe 35.7 ±7.98 63.2 ± 16.4 33.6 ± 1.62 31.4 ± 8.17 (μg · μl⁻¹ × 10⁻⁴) Renal *28.5± 4.8  1.66 ± 0.82 19.9 ± 6.4  0.58 ± 0.28 clearance Cu (μl · min⁻¹)Renal 0.25 ± 0.07 0.38 ± 0.15 0.46 ± 0.22 0.11 ± 0.03 clearance Fe (μl ·min⁻¹) Values shown as mean ± SEM. Asterisk indicates a significantdifference (P < 0.05) between diabetic animals receiving trientine anddiabetic animals receiving an equivalent volume of saline.

Metal content of cardiac tissue (Table 3): Wet heart weights in diabeticanimals were significantly less than those in nondiabetic animals whileheart/body weight ratios were increased. In some animals cardiac tissuewas also analyzed for Cu and Fe content. There was no significantdifference in content of copper between diabetic and nondiabetic animalsreceiving saline or trientine. Iron content of the non-diabetic animalsadministered saline was significantly greater than that of the diabeticanimals administered saline.

TABLE 3 Heart weight, heart weight/body weight ratios and trace metalcontent of heart tissue in diabetic versus nondiabetic animals. DIABETICNONDIABETIC Wet heart weight *0.78 ± 0.02 g 1.00 ± 0.02 g Heartweight/body *2.93 ± 0.05 mg · g⁻¹ 2.30 ± 0.03 mg · g⁻¹ weight Cu contentμg · g⁻¹ dry tissue Trientine treated  24.7 ± 1.5 27.1 ± 1.0 Salinetreated  21.3 ± 0.9 27.2 ± 0.7 Fe content μg · g⁻¹ dry tissue Trientinetreated   186 ± 46  235 ± 39 Saline treated  ^(†)180 ± 35  274 ± 30Diabetic animals: n = 5; nondiabetic animals: n = 10. Values shown asmean ± SEM. Asterisk indicates a significant difference (P < 0.05)between diabetic and non-diabetic animals. †indicates a significantdifference (P < 0.05) between diabetic and non-diabetic animalsreceiving saline.

Results from application of a mixed linear model to the experimentalanalysis (FIG. 35).

Copper: Diabetic rats excreted significantly higher levels of copperacross all dose levels. Baseline copper excretion was also significantlyhigher in diabetic rats compared to and prior to drug administration.The drug resulted in a significantly higher excretion of copper comparedto saline at all dose levels. There was no difference at baseline levelsbetween the drug and saline groups. The interaction effect for the modelwas significant at dose levels of 1.0 mg·kg⁻¹ and above. The presence ofa significant interaction term means that the influence of one effectvaries with the level of the other effect. Therefore, the outcome of asignificant interaction between the diabetes and drug factors isincreased copper excretion above the predicted additive effects of thesetwo factors.

Iron: Diabetic rats in the saline only group excreted significantlyhigher levels of iron at all dose levels. This resulted in all factorsin the model being significant across all dose levels.

In sum, the acute effect of intravenous trientine administration on thecardiovascular system and urinary excretion of copper and iron wasstudied in anesthetized, diabetic (6 weeks of diabetes, Streptozotocininduced) and nondiabetic rats. Animals were assigned to one of fourgroups: diabetic+trientine, diabetic+saline, nondiabetic+trientine,nondiabetic+saline, Drug, or an equivalent volume of saline, wasadministered hourly in doses of increasing strength (0.1, 1.0, 10, 100mg·kg⁻¹) and urine was collected throughout the experiment in 15 minaliquots. A terminal blood sample was taken and cardiac tissueharvested. Analysis of urine samples showed the following main points:

-   -   At all drug doses, diabetic and nondiabetic animals receiving        drug excreted more Cu (μg) than animals receiving an equivalent        volume of saline.    -   When analyzed per gram of bodyweight, diabetic animals excreted        significantly more copper (μg·gBW⁻¹) at each dose of trientine        than did nondiabetic animals. The same pattern was seen in        response to saline but the effect was not significant at every        dose.    -   At most doses, in diabetic animals iron excretion (μg) was        greater in animals administered saline than in those        administered drug. In nondiabetic animals there was no        difference between iron excretion in response to saline or        trientine administration.    -   Analysis per gram of body weight shows no difference between        iron excretion in nondiabetic and diabetic animals receiving        trientine. Diabetic animals receiving saline excrete more iron        per gram of bodyweight than nondiabetic animals receiving        saline.    -   Analysis of heart tissue showed no significant difference in        total copper content between diabetic and nondiabetic animals,        nor any effect of drug on cardiac content of iron and copper.    -   Renal clearance calculations showed a significant increase in        clearance of copper in diabetic animals receiving trientine        compared with diabetic animals receiving saline. The same trend        was seen in nondiabetic animals but the affect was not        significant. There was no affect of trientine on renal clearance        of iron.

Thus, there were no adverse cardiovascular effects were observed afteracute administration of trientine. Trientine treatment effectivelyincreases copper excretion in both diabetic and nondiabetic animals. Theexcretion of copper in urine following trientine administration isgreater per gram of bodyweight in diabetic than in nondiabetic animals.Iron excretion was not increased by trientine treatment in eitherdiabetic or nondiabetic animals.

Experiments relating to the efficacy of trientine to restore cardiacfunction in STZ diabetic rats were also carried out. As noted above,histological evidence from earlier studies showed that treatment withtrientine appears to protect the hearts of diabetic Wistar rats fromdevelopment of cardiac damage (diabetic cardiomyopathy), as judged byhistology. However, it was unknown whether this histological improvementtranslates into an improvement in cardiac function. One aim of thisstudy was to use an isolated-working-rodent heart model to comparecardiac function in trientine-treated and non-treated, STZ diabetic andnormal rats.

Male albino Wistar rats weighing 330-430 g were assigned to fourexperimental groups as follows:

Experimental groups Group Code N Treatment Group A STZ 8 Diabetes for 13weeks Group B STZ/D6 8 Diabetes for 13 weeks (Drug therapy week 7-13)Group C Sham 9 Non-diabetic controls Group D Sham/D7 11 Non-diabeticcontrols (Drug therapy week 7-13) STZ = Streptozotocin; D7 = trientinetreatment for 7 consecutive weeks commencing 6 weeks after the start ofthe experiment.

Diabetes was induced by intravenous streptozotocin (STZ; Sigma; St.Louis, Mo.). All rats were given a short inhalational anaesthetic(Induction: 5% halothane and 2 L/min oxygen, maintained on 2% halothaneand 2 L/min oxygen). Those in the two diabetic groups then received asingle intravenous bolus dose of STZ (57 mg/kg body weight) in 0.5 ml of0.9% saline administered via a tail vein. Non-diabetic sham-treatedanimals received an equivalent volume of 0.9% saline. Diabetic andnon-diabetic rats were housed in like-pairs and provided with freeaccess to normal rat chow (Diet 86 pellets; New Zealand Stock Feeds,Auckland, NZ) and deionized water ad libitum. Each cage had two waterbottles on it to ensure equal access to water or drug for each animal.Animals were housed at 21 degrees and 60% humidity in standard rat cageswith a sawdust floor that was changed daily.

Blood glucose was measured in tail-tip capillary blood samples(Advantage II, Roche Diagnostics, NZ Ltd). Sampling was performed on allgroups at the same time of the day. Blood glucose and body weight weremeasured on day 3 following STZ/saline injection and then weeklythroughout the study. Diabetes was confirmed by presence of polydipsia,polyuria and hyperglycemia (>11 mmol·L⁻¹).

In the drug treated diabetic group, trientine was prepared in thedrinking water for each cage at a concentration of 50 mg/L. Each animalconsumed about 260 ml water per day once diabetes was established, toyield a total drug dose per animal per day of ˜13 mg/kg. Thetrientine-containing drinking water was administered continuously fromthe start of week 7 until the animal was sacrificed at the end of week13. In the case of the Sham/D7 non-diabetic group that drank less waterper day than diabetic animals, the drug concentration in their drinkingwater was adjusted each week so that they consumed approximately thesame dose as the corresponding STZ/D7 group. At the time the drugstarted in the diabetic group the diabetic animals were expected to haveto have established cardiomyopathy, as shown by preliminary studies(data not shown) and confirmed in the literature. See Rodrigues B, etal., Diabetes 37(10):1358-64 (1988).

On the last day of the experiment, animals were anesthetized (5%halothane and 2 L·min⁻¹ O₂), and heparin (500 IU·kg⁻¹) (WeddelPharmaceutical Ltd., London) administered intravenously via tail vein. A2 ml blood sample was then taken from the inferior vena cava and theheart was then rapidly excised and immersed in ice-cold Krebs-Henseleitbicarbonate buffer to arrest contractile activity. Hearts were thenplaced in the isolated perfused working heart apparatus.

The aortic root of the heart was immediately ligated to the aorticcannula of the perfusion apparatus. Retrograde (Langendorff) perfusionat a hydrostatic pressure of 100 cm H₂O and at 37° C. was establishedand continued for 5 min while cannulation of the left atrium via thepulmonary vein was completed. The non-working (Langendorff) preparationwas then converted to the working heart model by switching the supply ofperfusate buffer from the aorta to the left atrium at a filling pressureof 10 cm H₂O. The left ventricle spontaneously ejected into the aorticcannula against a hydrostatic pressure (after-load) of 76 cmH₂O (55.9mmHg). The perfusion solution was Krebs-Henseleit bicarbonate buffer(mM: KCl 4.7, CaCl₂ 2.3, KH₂PO₄. 1.2, MgSO₄ 1.2, NaCl 118, and NaHCO₃25), pH 7.4 containing 11 mM glucose and it was continuously gassed with95% O₂:5% CO₂. The buffer was also continuously filtered in-line(initial 8 μm, following 0.4 μm cellulose acetate filters; Sartorius,Germany). The temperature of the entire perfusion apparatus wasmaintained by water jackets and buffer temperature was continuouslymonitored and adjusted to maintain hearts at 37° C. throughoutperfusion.

A modified 24 g plastic intravenous cannula (Becton Dickson, Utah, USA)was inserted into the left ventricle via the apex of the heart using thenormal introducer-needle. This cannula was subsequently attached to aSP844 piezo-electric pressure transducer (AD Instruments) tocontinuously monitor left ventricular pressure. Aortic pressure wascontinuously monitored through a side arm of the aortic cannula with apressure transducer (Statham Model P23XL, Gould Inc., CA, USA). Theheart was paced (Digitimer Ltd, Heredfordshire, England) at a rate of300 bpm by means of electrodes attached to the aortic and pulmonary veincannulae using supra-threshold voltages with pulses of 5-ms durationfrom the square wave generator.

Aortic flow was recorded by an in-line flow meter (Transonic T206,Ithaca, N.Y., USA) and coronary flow was measured by timed 30 seccollection of the coronary vein effluent at each time point step of theprotocol.

The working heart apparatus used was a variant of that originallydescribed by Neely, J R, et al., Am J Physiol 212:804-14 (1967). Themodified apparatus allowed measurements of cardiac function at differentpre-load pressures (FIG. 14 and FIG. 15). This was achieved byconstructing the apparatus so that the inflow height of the buffercoming to the heart could be altered through a series of graduated stepsin a reproducible manner. As in the case of the pre-load, the outflowtubing from the aorta could also be increased in height to provide aseries of defined after-load pressures. The after-load heights have beenconverted to mm Hg for presentation in the results which is in keepingwith published convention.

All data from the pressure transducers and flow probe were collected(Powerlab 16s data acquisition machine; AD Instruments, Australia). Thedata processing functions of this device were used to calculate thefirst derivative of the two pressure waves (ventricular and aortic). Thefinal cardiac function data available comprised:

Cardiac output*; aortic flow; coronary flow; peak leftventricular/aortic pressure developed; maximum rate of ventricularpressure development (+dP/dt)**; maximum rate of ventricular pressurerelaxation (−dP/dt)**; maximum rate of aortic pressure development(aortic +dP/dt); maximum rate of aortic relaxation (aortic −dP/dt).

[*Cardiac output (CO) is the amount of buffer pumped per unit time bythe heart and is comprised of buffer that is pumped out the aorta aswell as the buffer pumped into the coronary vessels. This is an overallindicator of cardiac function. **+dP/dt is the rate of change ofventricular (or aortic pressure) and correlates well with the strengthof the contraction of the ventricle (contractility). It can be used tocompare contractility abilities of different hearts when at the samepre-load (Textbook of Medical Physiology, Ed. A. Guyton. Saunderscompany 1986). −dP/dt is an accepted measurement of the rate ofrelaxation of the ventricle].

The experiment was divided into two parts, the first with fixedafter-load and variable pre-load the second, which immediately followedon from the first, with fixed pre-load and variable after-load.

Fixed After-load and changing Pre-load: After the initial cannulationwas completed, the heart was initially allowed to equilibrate for 6 minat 10 cm H₂O atrial filling pressure and 76 cm H₂O after-load. Duringthis period the left ventricular pressure transducer cannula wasinserted and the pacing unit started. Once the heart was stable, theatrial filling pressure was then reduced to 5 cm H₂O of water and thenprogressively increased in steps of 2.5 cmH₂O over a series of 7 stepsto a maximum of 20 cmH₂O. The pre-load was kept at each filling pressurefor 2 min, during which time the pressure trace could be observed tostabilize and the coronary flow was measured. On completion of thevariable pre-load experiment, the variable after-load portion of theexperiment was immediately commenced.

Fixed Pre-load and changing After-load: During this part of theexperiment the filling pressure (pre-load) was set at 10 cm H₂O and theafter-load was then increased from 76 cm H₂O (55.9 mm Hg) in steps of 8cm H₂O (5.88 mmHg); again each step was of 2 min duration. The maximumheight (after-load) to which each individual heart was ultimatelyexposed, was determined either by attainment of the maximal availableafter-load height of 145 cm H₂O (106.66 mm Hg), or the height at whichmeasured aortic flow became 0 ml/min. In the later situation, the heartwas considered to have “functionally failed.” To ensure that thisfailure was indeed functional and not due to other causes (e.g.,permanent ischaemic or valvular damage) all hearts were then returned tothe initial perfusion conditions (pre-load 10 cm H2O; after-load 75 cmH2O) for 4 minutes to confirm that pump function could be restored. Atthe end of this period the hearts were arrested with a retrogradeinfusion of 0.4 ml of cold KCL (24 mM). The atria and vascular remnantswere then excised, the heart blotted dry and weighed. The ventricleswere incised midway between the apex and atrioventricular sulcus.Measurements of the ventricular wall thickness were then made using amicro-caliper (Absolute Digimatic, Mitutoyo Corp, Japan).

Data from the Powerlab was extracted by averaging 1 min intervals fromthe stable part of the electronic trace generated from each step in theprotocol. The results from each group were then combined and analyzedfor differences between the groups for the various cardiac functionparameters (aortic flow, cardiac flow, MLVDP, LV or aortic +/−dP/dt).Differences between repeated observations at different pre-loadconditions were explored and contrasted between study group using amixed models approach to repeated measures (SAS v8.1, SAS Institute Inc,Cary N.C.). Missing random data were imputed using a maximum likelihoodapproach. Significant mean and interaction effects were further examinedusing the method of Tukey to maintain a pairwise 5% error rate for posthoc tests. All tests were two-tailed. Survival analysis was done usingProc Liftest (SAS V8.2). A one-way analysis of variance was used to testfor difference between groups in various weight parameters. Tukey'stests were used to compare each group with each other. In each graphunless otherwise stated. * indicates p<0.05=STZ v STZ/D7, #.p<0.05=STZ/D7 v Sham/D7.

Results showing that the weights of the animals at the end of theexperimental period are found in Table 4. Diabetic animals were about50% smaller than their corresponding age matched normals. A graph of thepercentage change in weight for each experimental group is found in FIG.5, wherein the arrow indicates the start of trientine treatment.

TABLE 4 Initial and final animal body weights (mean ± SD) Initial weightFinal weight Number (n) Treatment (g) (g) Group A* 9 STZ 361 ± 12 221 ±27 Group B* 8 STZ/D7 401 ± 33 290 ± 56 Group C* 8 Sham 361 ± 16 574 ± 50Group F 11 Sham/D7 357 ± 7  563 ± 17 *P < 0.05

Blood glucose values for the three groups of rats are presented in FIG.6. Generally, the presence of diabetes was established and confirmedwithin 3-5 days following the STZ injection, The Sham and Sham/D7control group remained normoglycemic throughout the experiment.Treatment with the drug made no difference to the blood glucose profile(p=ns) in either treated group compared to their respective appropriateuntreated comparison group.

Final heart weight and ventricular wall thickness measurements arepresented in Table 5. There was a small but significant improvement inthe “heart:body weight” ratio with treatment in the diabetic animals.There was a trend toward improved “ventricular wallthickness:bodyweight” ratio in treated diabetics compared to non-treatedbut this did not reach significance.

TABLE 5 Final heart weights (g) and per g of animal body Weight (BW)(mean ± SD) Left Ventricular Left Ventricular wall wall thickness Heartweight (g)/ thickness per BW Group Heart weight (g) BW (g) (mm) (mm)/(g)Sham 1.58 ± 0.13^(§) 0.0028 ± 0.0002^(§) 3.89 ± 0.38^(§) 0.0068 ±0.0009^(§) STZ/D7* 1.18 ± 0.24 0.0041 ± 0.0005 3.79 ± 0.52 0.0127 ±0.0027 STZ* 1.03 ± 0.17 0.0047 ± 0.0004 3.31 ± 0.39 0.0152 ± 0.0026Sham/D7 1.58 ± 0.05^(§) 0.0028 ± 0.0001^(§) 4.03 ± 0.1^(§) 0.0072 ±0.0003^(§) *P < 0.05 ^(§)= significant with the STZ and STZ/D7 groups p< 0.05

Part I results: The following graphs of FIGS. 7 to 12 represent cardiacperformance parameters of the animals (STZ diabetic; STZ diabetic+drug;and sham-treated controls) while undergoing increasing atrial fillingpressure (5-20 cmH₂O, pre-load) with a constant after-load of 75 cm H₂O.All results are mean±sem. In each graph for clarity unless otherwisestated, only significant differences related to the STZ/D7 the othergroups are shown:* indicates p<0.05 for STZ v STZ/D7, i# p<0.05 forSTZ/D7 v Sham/D7. Unless stated, STZ/D7 v Sham or Sham/D7 was notsignificant.

Cardiac output (FIG. 7) is the sum to the aortic flow (FIG. 10) and thecoronary flow as displayed in FIG. 8. Since the control hearts andexperimental groups have significantly different final weights, thecoronary flow is also presented (FIG. 9) as the flow normalized to heartweight (note that coronary flow is generally proportional to cardiacmuscle mass, and therefore to cardiac weight)

The first derivative of the pressure curve gives the rate of change inpressure development in the ventricle with each cardiac cycle and themaximum positive rate of change (+dP/dt) value is plotted in FIG. 11.The corresponding maximum rate of relaxation (−dP/dt) is in FIG. 12.Similar results showing improvement in cardiac function were found fromthe data derived from the aortic pressure cannula (results not shown).

Part II Results:

Under conditions for constant pre-load and increasing after-load theability of the hearts to cope with additional after-load work wasassessed. The plot of functional survival, that is the remaining numberof hearts at each after-load that still had an aortic output of greaterthan Oml/min is found in FIG. 13 and Table 6.

TABLE 6 Cardiac survival at each after-load pressure Number survivingPercentage functioning at each (aortic flow >0 mls/min) afterloadAfterload (mmHg) STZ STZ/D7 Sham Sham/D7 STZ STZ/D7 Sham Sham/D7 55.9 88 9 11 100% 100% 100% 100% 61.8 8 8 9 11 100% 100% 100% 100% 67.7 8 8 911 100% 100% 100% 100% 71.4 6 8 9 11 75% 100% 100% 100% 77.2 5 8 9 1163% 100% 100% 100% 83.1 4 8 9 11 50% 100% 100% 100% 88.3 3 7 9 11 38%88% 100% 100% 94.9 1 6 9 11 13% 75% 100% 100% 100.8 0 5 9 11 0% 63% 100%100% 106.7 0 1 9 9 0% 13% 100% 82%

In sum, for example,

-   -   Treatment with trientine had no obvious effect on blood glucose        concentrations in the two diabetic groups (as expected).    -   There was a small but significant improvement in the (heart        weight)/(body weight) ratio in the trientine-treated diabetic        group compared to that of the untreated diabetic group.    -   When the Pre-load was increased with the After-load held        constant, cardiac output was restored to Sham values. Both the        aortic and absolute coronary flows improved in the drug treated        group.    -   Indicators for ventricular contraction and relaxation were both        significantly improved in the drug treated group compared to        equivalent values in the untreated diabetic group. The        improvement restored function to such an extent that there was        no significant difference between the drug treated and the        sham-treated control groups.    -   The aortic transducer measures of pressure change also showed        improved function in the drug treated diabetic group compared to        the untreated diabetics (data not shown).    -   When after-load was increased in the presence of constant        pre-load, it was observed that the heart's ability to function        at higher after-loads was greatly improved in the drug treated        diabetic group compared to the untreated diabetic group. When        50% of the untreated diabetic hearts had failed, about 90% of        the trientine treated diabetic hearts were still functioning.    -   Compared to the untreated diabetic hearts, the response of the        drug treated diabetic hearts showed significant improvements in        several variables: cardiac output, aortic flow, coronary flow,        as well as improved ventricular contraction and relaxation        indices.    -   Drug treatment of normal animals had no adverse effects on        cardiac performance.

It is concluded that treatment of STZ diabetic rats with trientinedramatically improves several measures of cardiac function. It is alsoconcluded that administration of oral trientine for 7 weeks in Wistarrats with previously established diabetes of 6 weeks duration resultedin a global improvement in cardiac function. This improvement wasdemonstrated by improved contractile function (; +dP/dT) and a reductionin ventricular stiffness (−dP/dT). The overall ability of the Trientinetreated diabetic heart to tolerate increasing after-load was alsosubstantially improved.

Therapeutic formulations for use in the methods and preparation of thecompositions of the present invention can be prepared by any methodswell known in the art of pharmacy. See, for example, Gilman et al.(eds.) GOODMAN AND GILMAN'S: THE PHARMACOLOGICAL BASES OF THERAPEUTICS(8th ed.) Pergamon Press (1990); and Remington, THE SCIENCE OF PRACTICEAND PHARMACY, 20th Edition. (2001) Mack Publishing Co., Easton, Pa.;Avis et al. (eds.) (1993) PHARMACEUTICAL DOSAGE FORMS: PARENTERALMEDICATIONS Dekker, N.Y.; Lieberman et al. (eds.) (1990) PHARMACEUTICALDOSAGE FORMS: TABLETS Dekker, N.Y.; and Lieberman et al. (eds.) (1990)PHARMACEUTICAL DOSAGE FORMS: DISPERSE SYSTEMS Dekker, N.Y. Dosage formsuseful herein include any appropriate dosage form well known in the artto be suitable for pharmaceutical formulation of compounds suitable foradministration to mammals particularly humans, particularly (althoughnot solely) those suitable for stabilization in solution of therapeuticcompounds for administration to mammals preferably humans. The dosageforms of the invention thus include any appropriate dosage form nowknown or later discovered in the art to be suitable for pharmaceuticalformulation of compounds suitable for administration to mammalsparticularly humans, particularly (although not solely) those suitablefor stabilization in solution of compounds for administration to mammalspreferably humans. One example is oral delivery forms of tablet,capsule, lozenge, or the like form, or any liquid form such as syrups,aqueous solutions, emulsion and the like, capable of protecting thecompound from degradation prior to eliciting an effect, for example, inthe alimentary canal if an oral dosage form. Examples of dosage formsfor transdermal delivery include transdermal patches, transdermalbandages, and the like. Included within the topical dosage forms are anylotion, stick, spray, ointment, paste, cream, gel, etc., whether applieddirectly to the skin or via an intermediary such as a pad, patch or thelike. Examples of dosage forms for suppository delivery include anysolid or other dosage form to be inserted into a bodily orifice(particularly those inserted rectally, vaginally and urethrally).Examples of dosage units for transmucosal delivery include depositories,solutions for enemas, pessaries, tampons, creams, gels, pastes, foams,nebulised solutions, powders and similar formulations containing inaddition to the active ingredients such carriers as are known in the artto be appropriate. Examples of dosage units for depot administrationinclude pellets or small cylinders of active agent or solid formswherein the active agent is entrapped in a matrix of biodegradablepolymers, microemulsions, liposomes or is microencapsulated. Examples ofimplantable infusion devices include any solid form in which the activeagent is encapsulated within or dispersed throughout a biodegradablepolymer or synthetic, polymer such as silicone, silicone rubber,silastic or similar polymer. Alternatively dosage forms for infusiondevices may employ liposome delivery systems.

Depending on the disease to be treated and the subject's condition, thecompounds of the present invention may be administered by oral,parenteral (for example, intramuscular, intraperitoneal, intravenous,ICV, intracisternal injection or infusion, subcutaneous injection, orimplant), by inhalation spray, nasal, vaginal, rectal, sublingual, ortopical routes of administration and may be formulated, alone ortogether, in suitable dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and vehiclesappropriate for each route of administration. The pharmaceuticalcomposition and method of the present invention may further compriseother therapeutically active compounds as noted herein which are usuallyapplied in the treatment of the above mentioned conditions.

In the treatment or prevention of conditions which require coppermodulation an appropriate dosage level will generally be about 0.001 to100 mg per kg patient body weight per day which can be administered insingle or multiple doses. Preferably, the dosage level will be about0.01 to about 25 mg/kg per day; more preferably about 0.05 to about 10mg/kg per day. A suitable dosage level may be about 0.01 to 25 mg/kg perday, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg per day.Within this range the dosage may be about 0.005 to about 0.05, 0.05 to0.5 or 0.5 to 5 mg/kg per day. For oral administration, the compositionsare preferably provided in the form of tablets containing about 1 to1000 milligrams of the active ingredient, particularly about 1, 5, 10,15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800,900, and 1000 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. The compounds maybe administered on a regimen of 1 to 4 times per day, preferably once ortwice per day.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the host undergoing therapy.

The compounds of the present invention can be combined with othercompounds having related utilities to prevent and treat tissue damage orexcess tissue copper.

All patents, publications, scientific articles, web sites, and otherdocuments and materials referenced or mentioned herein are indicative ofthe levels of skill of those skilled in the art to which the inventionpertains, and each such referenced document and material is herebyincorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such patents, publications, scientific articles,web sites, electronically available information, and other referencedmaterials or documents.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “consisting essentially of”, and“consisting of” may be replaced with either of the other two terms inthe specification. As used herein the term “and/or” means both “and” and“or”. As used herein the addition of “(s)” as part of a word embracedboth the singular and plural of that word. Also, the terms “comprising”,“including”, containing”, etc. are to be read expansively and withoutlimitation. The methods and processes illustratively described hereinsuitably may be practiced in differing orders of steps, and that theyare not necessarily restricted to the orders of steps indicated hereinor in the claims. It is also that as used herein and in the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise. Under no circumstancesmay the patent be interpreted to be limited to the specific examples orembodiments or methods specifically disclosed herein. Under nocircumstances may the patent be interpreted to be limited by anystatement made by any Examiner or any other official or employee of thePatent and Trademark Office unless such statement is specifically andwithout qualification or reservation expressly adopted in a responsivewriting by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. (canceled)
 2. A method of treating tissue damage relating to coppervalues content in a mammal, said tissue being selected from myocardialtissue, kidney tissue, eye tissue, nerve tissue, and vascular tissue,the method comprising administering to said mammal in need thereof, anamount of an agent or agents effective to lower the copper (II) valuescontent in one or more of said tissues and thereby treat said damage,wherein said mammal does not have Wilson's Disease or diabetes mellitus,and wherein the agent(s) is (are) selected from the group consisting of:trientine; ethylenediaminetetraacetic acid (EDTA);diethylenetriaminetetraacetic acid (DPTA); 2,2,2 tetraminetetrahydrochloride (TETA); 2,3,2 tetramine tetrahydrochloride;5,7,7′12,14,14′ hexaxmethyl-1,4,8,11 tetraazacyclotretradecane (CyclamS); 3-7-Diazanonan-1,9-diamin (BE 6184);1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid;1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;4,11-bis(N,N-diethyl-amidomethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;4,11-bis(amidoethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;melatonin; N,N′-diethyldithiocarbamate; bathocuproinedisulfonic acid;bathocuprinedisulfonate; trimetazidine; triethylene tetraminetetrahydrochloride; 2,3,2-tetraamine; 1,10-orthophenanthroline;3,4-Dihydroxybenzoic acid; 2,2′-bicinchinonic acid; diamsar; 3,4′,5,trihydroxystilbene (resveratrol); mercaptodextran; o-phenanthroline;disulfuram (antabuse); sar; diethylene triamine pentaacetic acid; andcalcium trisodium diethylenetriaminepentaacetate.
 3. The method of claim2 wherein the damage relates to elevated copper (II) content.
 4. Themethod of claim 2 wherein the mammal is a human.
 5. The method of claim4 wherein the human has an elevated copper (II) values content.
 6. Themethod of claim 4 further comprising making at least one copper valuesstatus determination in said human.
 7. The method of claim 4 wherein thecopper (II) values content in said human is lowered.
 8. The method ofclaim 2 wherein said agent is a trientine.
 9. The method of claim 8wherein said trientine is trientine hydrochloride and is administered atdosages or a dosage to provide, if parenteral, at least about 120 mg/dayin said a human, and if oral, at least about 600 or at least about 1200mg/day in said human.
 10. The method of claim 2 wherein said damage isfrom any one or more of (i) cardiomyopathy or -myocarditis; (ii)atheromatous disorders of the major blood vessels; (iii) disorders ofsmall blood vessels in myocardial tissue, kidney tissue, eye tissue,nerve tissue, and vascular tissue; and/or; (iv) non-fatal plaque ruptureof atheromatous lesions of major blood vessels.
 11. A method of treatinga mammalian subject at risk of developing, with suspected or with actualtissue disease relating to copper values content in the myocardium,kidney, eye, nervous system, and/or vascular system, which methodcomprises the step of administering to the subject one or more agentscapable of decreasing the copper (II) values content, whereby saidtissue disease in said subject is improved by decreasing the copper (II)values content of the tissue, and wherein the subject does not haveWilson's Disease or diabetes mellitus, and wherein the agent(s) is (are)selected from the group consisting of: trientine;ethylenediaminetetraacetic acid (EDTA); diethylenetriaminetetraaceticacid (DPTA); 2,2,2 tetramine tetrahydrochloride (TETA); 2,3,2 tetraminetetrahydrochloride; 5,7,7′12,14,14′ hexaxmethyl-1,4,8,11tetraazacyclotretradecane (Cyclam S); 3-7-Diazanonan-1,9-diamin (BE6184); 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid;1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;4,11-bis(N,N-diethyl-amidomethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;4,11-bis(amidoethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane;melatonin; N,N′-diethyldithiocarbamate; bathocuproinedisulfonic acid;bathocuprinedisulfonate; trimetazidine; triethylene tetraminetetrahydrochloride; 2,3,2-tetraamine; 1,10-orthophenanthroline;3,4-Dihydroxybenzoic acid; 2,2′-bicinchinonic acid; diamsar; 3,4′,5,trihydroxystilbene (resveratrol); mercaptodextran; o-phenanthroline;disulfuram (antabuse); sar; diethylene triamine pentaacetic acid; andcalcium trisodium diethylenetriaminepentaacetate.
 12. The method ofclaim 11 wherein the damage relates to elevated copper (II) content. 13.The method of claim 11 wherein the mammal has an elevated copper (II)values content.
 14. The method of claim 11 wherein the copper (II)values content in said human is lowered.
 15. The method of claim 9wherein said damage is from any one or more of (i) cardiomyopathy or-myocarditis; (ii) atheromatous disorders of the major blood vessels;(iii) disorders of small blood vessels in myocardial tissue, kidneytissue, eye tissue, nerve tissue, and vascular tissue; and/or; (iv)non-fatal plaque rupture of atheromatous lesions of major blood vessels.